Method for Producing Alpha-substituted Cysteine or Salt Thereof or Synthetic Intermediate of Alpha-substituted Cysteine

ABSTRACT

According to the present invention, it becomes possible to perform a process for converting into an α-substituted cysteine represented by general formula (1) or a salt thereof at low cost and on an industrial scale by employing a process that is routed through a compound represented by general formula (3) to a compound represented by general formula (6). Particularly, by employing a process that is routed through a compound represented by general formula (7-2), it becomes possible to detach a tert-butyl protection group in a simple manner and to produce the compound represented by general formula (1) with high purity. Furthermore, by employing a process that is routed through tert-butylthiomethanol or a process that is routed through a compound represented by general formula (9), it becomes possible to produce a compound represented by general formula (2) without generating bischloromethylether that is an oncogenic substance. In the production of an α-substituted-D-cysteine or a salt thereof, it becomes possible to perform a process for converting the compound represented by general formula (2) into a compound represented by general formula (3S) in one step by allowing an enzyme or the like to act on the compound represented by general formula (2).

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 14/888,576, which isthe U.S. national phase of PCT/JP2014/062115 filed May 1, 2014, based onJP 2013-097172 filed May 2, 2013, the entire respective disclosures ofwhich are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method for producing an α-substitutedcysteine or a salt thereof, which is useful as an intermediate forpharmaceuticals and the like, or an intermediate for synthesis of anα-substituted cysteine.

BACKGROUND ART

Among α-substituted cysteines, optically active α-substituted cysteinesare especially useful as intermediates for pharmaceuticals. Variousmethods are conventionally known as methods for producing α-substitutedcysteines and salts thereof (for example, Patent Document 1).

However, the conventional methods have been impractical since stableproduction in an industrial scale has been difficult because of, forexample, requirement of a low-temperature reaction using an expensivebase such as butyllithium, and/or requirement of many laborious stepsusing a large amount of expensive reagents.

As a method that does not require an expensive reagent or alow-temperature reaction unlike the method in Patent Document 1, PatentDocument 2 describes a method for producing α-methyl-D-cysteine in whichracemic N-carbamoyl-α-methylcysteine is subjected to D-isomer-specificcyclization by hydantoinase to produce D-5-methyl-5-thiomethylhydantoin,followed by hydrolysis, decarbamoylation, and sulfur atom deprotection(elimination of the tert-butyl group) of theD-5-methyl-5-thiomethylhydantoin. This α-methyl-D-cysteine is anoptically active α-substituted cysteine, and useful as an intermediatefor a therapeutic agent for hyperferremia.

Patent Document 3 describes a method for obtaining benzyl-protectedα-methyl-D-cysteine by allowing Bacillus licheniformis protease to acton a diester having benzyl-protected thiol to provide an (S)-monoester,and then converting the isocyanate group generated by Curtiusrearrangement to an amino group.

Non-patent Document 1 discloses a method for obtainingtert-butyl-protected α-methyl-D-cysteine, wherein a diester protectedwith a tert-butyl group instead of a benzyl group is subjected to(R)-monoesterification using pig liver esterase, and then to tert-butylesterification of the carboxyl group and hydrolysis of the methyl esterto obtain an (S)-monoester, followed by converting the isocyanate groupgenerated by Curtius rearrangement to a carbamate-protected amino group,and then deprotecting the carbamate protection.

Non-patent Document 2 describes a production method in which thetert-butyl-protected diester described in Non-patent Document 1,tert-butylthiochloromethane, and methyl dialkylmalonate are reacted inthe presence of a base. The document also describes a method forproducing tert-butylthiochloromethane, in which tert-butyl mercaptan,paraformaldehyde, and hydrogen chloride are reacted in dichloromethane.It is known that, in this process, contacting of formaldehyde withhydrogen chloride causes generation of bis-chloromethylether (forexample, Non-patent Document 3).

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: WO 2001/072702-   Patent Document 2: WO 2003/106689-   Patent Document 3: WO 2007/106022

Non-Patent Documents

-   Non-patent Document 1: Journal of Organic Chemistry (2003),    68(13), p. 5403-5406-   Non-patent Document 2: Synlett (2010), (19), p. 2941-2943-   Non-patent Document 3: Chemische Berichte, 88, 1737 (1955)

SUMMARY OF THE INVENTION

In the method described in Patent Document 2, a long reaction time isrequired for each of the steps of hydrolysis, decarbamoylation, andsulfur atom deprotection of hydantoin, so that the production efficiencyis low. Moreover, since optical resolution is used as the method forobtaining the D-isomer, the N-carbamoyl-α-methylcysteine to be subjectedto the optical resolution needs to be produced in an amount of at leasttwo molar equivalents with respect to the amount of interest. Inaddition, a disposal process is required for the unnecessary L-isomer.

In the method described in Patent Document 3, the (S)-monoester can beobtained from the diester at a theoretical yield of 100% (that is, thebenzyl-protected α-methyl-D-cysteine can also be obtained from thediester at a theoretical yield of 100%). Therefore, the above-mentionedproblem in Patent Document 2 does not occur. However, in order to obtainα-methyl-D-cysteine, the benzyl group on the sulfur atom, which is aprotecting group, needs to be eliminated. In general, Birch reductionusing liquid ammonia and sodium metal is employed for elimination of abenzyl group. In this method, very low temperature conditions arerequired, and sodium metal, which is highly ignitable, needs to be used,so that the method is not suitable for industrial production. Moreover,since the deprotected α-methyl-D-cysteine is a compound whose extractionwith an organic solvent is difficult, removal of the metal saltgenerated during the reaction is difficult.

In the method described in Non-patent Document 1, the elimination of thetert-butyl group is achieved by heating in the presence of hydrochloricacid. Therefore, the problem in Patent Document 3 does not occur.However, since the operation of conversion of the (R)-monoester to the(S)-monoester is laborious, the method is not suitable for industrialproduction. Moreover, the optical purity of the (S)-monoester obtainedby this method depends on the (R)-monoester, and the optical purity ofthe (R)-monoester is 91% e.e. Thus, the optical purity is insufficientfor providing an intermediate for production of a pharmaceutical.

In cases where a tert-butyl-protected diester is produced by the methoddescribed in Non-patent Document 2, bis-chloromethylether, which ishighly carcinogenic and, especially in Japan, designated as a substancewhose production is prohibited under Article 55 of the Safety and HealthAct, may be generated.

The present invention was made in view of the above circumstances, andaims to provide a practical method that enables simpler, quicker, andsafer production of an α-substituted cysteine or a salt thereof which isuseful as an intermediate for pharmaceuticals and the like using aneasily available and inexpensive material, which method is applicable tostable, industrial-scale production of the α-substituted cysteine or thesalt thereof.

As a result of intensive study on a method for producing anα-substituted cysteine or a synthetic intermediate thereof, the presentinventors discovered that, by allowing a reaction to proceed through2-oxo-1,3-thiazolidine-4-carboxylic acid, elimination of a tert-butylprotecting group, which has conventionally required a very long reactiontime, can be achieved in a short time. The present inventors alsodiscovered that, by employing a process of synthesis fromtert-butylthiomethanol or dialkyl 2-[(tert-butylthio)methyl]malonate,dialkyl 2-(tert-butylthio)methyl-2-substituted malonate can be safelyproduced without generating bis-chloromethylether, which iscarcinogenic.

In addition, the present inventors discovered that, in production of anα-substituted-D-cysteine, which is especially useful as an intermediatefor a therapeutic agent for hyperferremia, the process of converting adialkyl 2-[(tert-butylthio)methyl]-2-substituted malonate to an alkyl(S)-2-[(tert-butylthio)methyl]-2-substituted malonate, which has beenlaborious, can be carried out in one step by enzymatic reaction and thelike.

That is, the present invention relates to the following.

[1] A method for producing an α-substituted cysteine represented byGeneral Formula (1):

(wherein R² represents a C₁-C₄ alkyl group) or a salt thereof, themethod comprising the steps of:

(i) allowing a base or an acid; or an enzyme having an activity tohydrolyze an ester group, a cell having an ability to produce theenzyme, a processed product of the cell, and/or a culture liquidcontaining the enzyme obtained by culturing the cell; to act on acompound represented by General Formula (2):

(wherein each R¹ independently represents a C₁-C₁₀ alkyl group which isoptionally substituted, a C₇-C₂₀ aralkyl group which is optionallysubstituted, or a C₆-C₁₂ aryl group which is optionally substituted, andR² represents a C₁-C₄ alkyl group), to obtain a compound represented byGeneral Formula (3):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (2));

(ii) allowing a condensing agent or an acid halogenating agent to act onthe compound represented by the General Formula (3), to obtain acompound represented by General Formula (4):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (2); X represents —OP(O)(OPh)₂, —OP(O)(OEt)₂, —OC(O)OR³, or ahalogen atom; and R³ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted);

(iii) allowing an azidation agent to act on the compound represented bythe General Formula (4), to obtain a compound represented by GeneralFormula (5):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (2));

(iv) converting, by Curtius rearrangement reaction, the compoundrepresented by the General Formula (5), to obtain a compound representedby General Formula (6):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (2)); and

(v) subjecting the compound represented by the General Formula (6) to aprocess of converting the isocyanate group to an amino group, a processof hydrolyzing the ester group, and a process of removing the tert-butylgroup by action of an acid.

[2] The method for producing an α-substituted cysteine or a salt thereofaccording to [1], wherein

in the Step (i), the enzyme is

-   -   (A) a protein comprising the amino acid sequence of SEQ ID NO:        4, 6, 8, 10, 12, 14, 16, or 18;    -   (B) a protein having an identity of not less than 35% to the        amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or        18, and having an activity to hydrolyze the compound represented        by the General Formula (2) for conversion into a compound        represented by General Formula (3S):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (3)), which is a compound represented by the General Formula (3)and having an (S)-configuration; or

-   -   (C) a protein comprising the amino acid sequence of SEQ ID NO:        4, 6, 8, 10, 12, 14, 16, or 18 in which one or several amino        acids are deleted, substituted, and/or added, and having an        activity to hydrolyze the compound represented by the General        Formula (2) for conversion into a compound represented by        General Formula (3S), which is a compound represented by the        General Formula (3) and having an (S)-configuration; and

the α-substituted cysteine represented by the General Formula (1) is anα-substituted cysteine represented by the following General Formula (1S)having an (S)-configuration:

(wherein R² has the same meaning as R² in the General Formula (1)).[3] A method for producing a compound represented by General Formula(3S):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² represents aC₁-C₄ alkyl group),which is a compound represented by the General Formula (3):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (3S))and having an (S)-configuration, the method comprising the step of:

(i) allowing an enzyme having an activity to hydrolyze an ester group, acell having an ability to produce the enzyme, a processed product of thecell, and/or a culture liquid containing the enzyme obtained byculturing the cell, to act on a compound represented by General Formula(2):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (3S)).[4] The method for producing a compound represented by the GeneralFormula (3S) according to [3], wherein the enzyme is

(A) a protein comprising the amino acid sequence of SEQ ID NO: 4, 6, 8,10, 12, 14, 16, or 18;

(B) a protein having an identity of not less than 35% to the amino acidsequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18, and having anactivity to hydrolyze the compound represented by the General Formula(2) for conversion into the compound represented by the General Formula(3S) having the (S)-configuration; or

(C) a protein comprising the amino acid sequence of SEQ ID NO: 4, 6, 8,10, 12, 14, 16, or 18 in which one or several amino acids are deleted,substituted, and/or added, and having an activity to hydrolyze thecompound represented by the General Formula (2) for conversion into thecompound represented by the General Formula (3S) having the(S)-configuration.

[5] A method for producing a compound represented by General Formula(4-1):

(wherein R¹ and R³ each independently represent a C₁-C₁₀ alkyl groupwhich is optionally substituted, a C₇-C₂₀ aralkyl group which isoptionally substituted, or a C₆-C₁₂ aryl group which is optionallysubstituted, and R² represents a C₁-C₄ alkyl group), the methodcomprising the step of:

(ii) allowing a chloroformic ester to act on a compound represented byGeneral Formula (3):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (4-1)).[6] A method for producing a compound represented by General Formula(5):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² represents aC₁-C₄ alkyl group),the method comprising the step of:

(iii) allowing a metal azide to act on a compound represented by GeneralFormula (4-1):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (5), and R³ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted).[7] A method for producing a compound represented by General Formula(6):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² represents aC₁-C₄ alkyl group),the method comprising the step of:

(iv) converting, by Curtius rearrangement reaction, a compoundrepresented by General Formula (5):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (6)).[8] A method for producing an α-substituted cysteine represented byGeneral Formula (1):

(wherein R² represents a C₁-C₄ alkyl group)or a salt thereof, the method comprising the step of:

(v) subjecting a compound represented by General Formula (6):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² has the samemeaning as R² in the General Formula (1)) to a process of converting theisocyanate group to an amino group, a process of hydrolyzing the estergroup, and a process of removing the tert-butyl group by action of anacid.[9] The method for producing an α-substituted cysteine represented byGeneral Formula (1):

(wherein R² represents a C₁-C₄ alkyl group)or a salt thereof according to [8], wherein the step (v) comprises thesteps of:

(vi-1) allowing an acid to act on a compound represented by GeneralFormula (6):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (6)) to construct a thiazolidinone ring, for conversion into acompound represented by General Formula (7-1):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (6));

(vi-2) allowing an acid or a base to act on the compound represented bythe General Formula (7-1) to hydrolyze the ester group, to obtain acompound represented by General Formula (7-2):

(wherein R² has the same meaning as R² in the General Formula (7-1));and

(vii) allowing an acid or a base to act on the compound represented bythe General Formula (7-2) to open the thiazolidinone ring, to producethe α-substituted cysteine represented by the General Formula (1) or thesalt thereof.

[10] A method for producing a compound represented by General Formula(7-1):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² represents aC₁-C₄ alkyl group),the method comprising the step of:

(vi-1) allowing an acid to act on a compound represented by GeneralFormula (6):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (7-1))to construct a thiazolidinone ring.[11] A method for producing a compound represented by General Formula(7-2):

(wherein R² represents a C₁-C₄ alkyl group),the method comprising the step of:

(vi-2) allowing an acid or a base to act on a compound represented byGeneral Formula (7-1):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² has the samemeaning as R² in the General Formula (7-2)) to hydrolyze the estergroup.[12] A method for producing an α-substituted cysteine represented byGeneral Formula (1):

(wherein R² represents a C₁-C₄ alkyl group)or a salt thereof, the method comprising the steps of:

(vii) allowing an acid or a base to act on a compound represented byGeneral Formula (7-2):

(wherein R² has the same meaning as R² in the General Formula (1))to open the thiazolidinone ring[13] A compound represented by General Formula (4-1):

(wherein R¹ and R³ each independently represent a C₁-C₁₀ alkyl groupwhich is optionally substituted, a C₇-C₂₀ aralkyl group which isoptionally substituted, or a C₆-C₁₂ aryl group which is optionallysubstituted, and R² represents a C₁-C₄ alkyl group).[14] A compound represented by General Formula (5):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² represents aC₁-C₄ alkyl group).[15] A compound represented by General Formula (6):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² represents aC₁-C₄ alkyl group).[16] A compound represented by General Formula (7S):

(wherein R² represents a C₁-C₄ alkyl group, and R⁵ represents a hydrogenatom, or a C₁-C₁₀ alkyl group which is optionally substituted, a C₇-C₂₀aralkyl group which is optionally substituted, or a C₆-C₁₂ aryl groupwhich is optionally substituted).[17] A method for producing a compound represented by General Formula(2):

(wherein each R¹ independently represents a C₁-C₁₀ alkyl group which isoptionally substituted, a C₇-C₂₀ aralkyl group which is optionallysubstituted, or a C₆-C₁₂ aryl group which is optionally substituted, andR² represents a C₁-C₄ alkyl group), the method comprising the step of:

(xi) allowing an alkylating agent to act on a compound represented byGeneral Formula (9):

(wherein R¹ has the same meaning as R¹ in the General Formula (2)) inthe presence of a base.[18] A method for producing a compound represented by General Formula(2):

(wherein each R¹ independently represents a C₁-C₁₀ alkyl group which isoptionally substituted, a C₇-C₂₀ aralkyl group which is optionallysubstituted, or a C₆-C₁₂ aryl group which is optionally substituted, andR² represents a C₁-C₄ alkyl group), the method comprising the steps of:

(viii) reacting tert-butyl mercaptan with formaldehyde to obtaintert-butylthiomethanol;

(ix) reacting tert-butylthiomethanol with a chlorinating agent in thepresence of a base to obtain tert-butylthiochloromethane; and

(x) allowing tert-butylthiochloromethane to act on a compoundrepresented by General Formula (8):

(wherein R¹ has the same meaning as R¹ in the General Formula (2), andR² represents a C₁-C₄ alkyl group) in the presence of a base, to obtainthe compound represented by the General Formula (2).

Effects of the Invention

According to the production method of the present invention, anα-substituted cysteine or a salt thereof can be simply, quickly, andsafely produced using an easily available and inexpensive material, andstable, industrial-scale production of the α-substituted cysteine or thesalt thereof can be achieved.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing an embodiment of the production method ofthe present invention.

FIG. 2 shows the result of HPLC analysis of the optical purity of thereaction product obtained by allowing an enzyme derived from theBacillus subtilis IFO3108 strain to act on diethyl2-(tert-butylthio)methyl-2-methylmalonate (Example 12). The upper panelshows the result of the reaction with the enzyme derived from theBacillus subtilis IFO3108 strain, and the lower panel shows the resultof analysis of the optical purity of a racemic sample which was carriedout as a control experiment.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Method for Producing α-Substituted Cysteine Represented by GeneralFormula (1) or Salt Thereof]

The method for producing an α-substituted cysteine represented by theGeneral Formula (1) shown below or a salt thereof of the presentinvention comprises the step (i) and the subsequent steps (ii), (iii),and (iv). The production method of the present invention preferablycomprises, before the step (i), the step (xi), or the steps (viii),(xi), and (x). The production method of the present invention preferablycomprises, after the step (iv), the step (v), especially preferably thesteps (vi-1), (vi-2), and (vii).

General Formula (1)

(wherein R² represents a C₁-C₄ alkyl group)<Step (i)>

First, the step (i) is described below.

The step (i) is a step of allowing a base or an acid; or an enzymehaving an activity to hydrolyze an ester group, a cell having an abilityto produce the enzyme, a processed product of the cell, and/or a cultureliquid containing the enzyme obtained by culturing the cell (the “enzymehaving an activity to hydrolyze an ester group, cell having an abilityto produce the enzyme, processed product of the cell, and/or cultureliquid containing the enzyme obtained by culturing the cell” may behereinafter referred to as “enzyme and/or the like of the presentinvention”); to act on a compound represented by General Formula (2):

(wherein each R¹ independently represents a C₁-C₁₀ alkyl group which isoptionally substituted, a C₇-C₂₀ aralkyl group which is optionallysubstituted, or a C₆-C₁₂ aryl group which is optionally substituted, andR² represents a C₁-C₄ alkyl group), to obtain a compound represented byGeneral Formula (3):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (2)).

The compound represented by General Formula (2) can be produced, asdescribed in Non-patent Document 2, by reactingtert-butylthiochloromethane and dialkyl methyl malonate in the presenceof a base. This document also describes a method for producingtert-butylthiochloromethane, in which tert-butyl mercaptan,paraformaldehyde, and hydrogen chloride are reacted in dichloromethane.However, according to Non-patent Document 3, contacting of formaldehydewith hydrogen chloride may cause generation of bis-chloromethylether,which is highly carcinogenic. Thus, this production method isunfavorable from the viewpoint of health of the operator.

Accordingly, the production method for the compound represented byGeneral Formula (2) is preferably a production method comprising thesteps (viii)-(x), in which tert-butylthiochloromethane is producedwithout bringing formaldehyde into contact with hydrogen chloride, orthe step (xi), in which tert-butylthiochloromethane is not used. Thesteps (viii)-(xi) are described later.

In the compound represented by General Formula (2), examples of thealkyl group of the “C₁-C₁₀ alkyl group which is optionally substituted”as R¹ include C₁-C₁₀ linear, branched, or cyclic alkyl groups such asmethyl, ethyl, isopropyl, n-propyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl,cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Examples of thesubstituent optionally contained in the alkyl group include C₁-C₆ alkoxygroups such as methoxy and ethoxy; halogen atoms such as a fluorineatom, chlorine atom, bromine atom, and iodine atom; and nitro. Thenumber of substituents is not limited, and, in cases where there are twoor more substituents in the alkyl group, the substituents may be of thesame type or different types.

In the compound represented by General Formula (2), examples of thearalkyl group of the “C₇-C₂₀ aralkyl group which is optionallysubstituted” as R¹ include benzyl, 2-phenylethyl, 1-phenylethyl,1-phenylpropyl, 2-phenylpropyl, and 3-phenylpropyl. Examples of thesubstituent optionally contained in the aralkyl group include C₁-C₆alkyl groups such as methyl, ethyl, and isopropyl; C₁-C₆ alkoxy groupssuch as methoxy and ethoxy; halogen atoms such as a fluorine atom,chlorine atom, bromine atom, and iodine atom; and nitro. The number ofsubstituents is not limited, and, in cases where there are two or moresubstituents in the aralkyl group, the substituents may be of the sametype or different types.

In the compound represented by General Formula (2), examples of the arylgroup of the “C₆-C₁₂ aryl group which is optionally substituted” as R¹include phenyl, 1-naphthyl, and 2-naphthyl. Examples of the substituentoptionally contained in the aryl group include C₁-C₆ alkyl groups suchas methyl, ethyl, and isopropyl; C₁-C₆ alkoxy groups such as methoxy andethoxy; halogen atoms such as a fluorine atom, chlorine atom, bromineatom, and iodine atom; and nitro. The number of substituents is notlimited, and, in cases where there are two or more substituents in thearyl group, the substituents may be of the same type or different types.

Among the groups described above, R¹ is preferably a C₁-C₁₀unsubstituted linear or branched alkyl group such as methyl, ethyl,isopropyl, n-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,isopentyl, tert-pentyl, neopentyl, or n-hexyl from the viewpoint oftheir availability, more preferably a C₁-C₄ unsubstituted linear orbranched alkyl group such as methyl, ethyl, isopropyl, n-propyl,n-butyl, isobutyl, sec-butyl, or tert-butyl from the viewpoint of theirinexpensiveness, still more preferably a C_(r) C₄ unsubstituted linearalkyl group such as methyl, ethyl, n-propyl, or n-butyl from theviewpoint of their reactivity with acids, bases and enzymes. Inparticular, in cases where an enzyme is used in the step (i), thesmaller the structure of R¹, the better it is incorporated into thereaction site of the enzyme, leading to a higher reaction rate. Thus,methyl or ethyl is most preferred.

In the compound represented by General Formula (2), the two R¹s are notnecessarily the same, and may be different from each other. In caseswhere the R¹s are different from each other, two enantiomers, the(R)-isomer and the (S)-isomer, are present as the compound representedby the General Formula (2). The mixing ratio between these enantiomersis not limited.

In the compound represented by General Formula (2), R², that is, theC₁-C₄ alkyl group, needs to be the same as R² in the compound ofinterest represented by General Formula (1). Examples of R² includemethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, andtert-butyl. From the viewpoint of ease of production, C₁-C₄unsubstituted linear alkyl groups such as methyl, ethyl, n-propyl, andn-butyl are more preferred. In particular, in cases where the enzymeand/or the like of the present invention is used, the smaller thestructure of R², the better it is incorporated into the reaction site ofthe enzyme, leading to a higher reaction rate. Thus, methyl or ethyl isstill more preferred. R² needs to be methyl in cases whereα-methyl-D-cysteine, which is useful as an intermediate for atherapeutic agent for hyperferremia, is produced among thelater-described α-substituted cysteines represented by General Formula(1) and salts thereof.

Examples of the compound represented by General Formula (2) include thefollowing compounds.

Among the compounds described above, the following compounds arepreferred as the compound represented by General Formula (2).

By allowing a base or an acid; or an enzyme having an activity tohydrolyze an ester group, a cell having an ability to produce theenzyme, a processed product of the cell, and/or a culture liquidcontaining the enzyme obtained by culturing the cell; to act on acompound represented by General Formula (2) to perform hydrolysis, acompound represented by General Formula (3) can be obtained.

In the compound represented by General Formula (3), R¹ and R² have thesame meanings as R¹ and R² in the above-mentioned General Formula (2).

Examples of the compound represented by General Formula (3) include thefollowing compounds.

Among the compounds described above, the following compounds arepreferred as the compound represented by General Formula (3).

The compound represented by General Formula (3) may be a chiral compoundwhich selectively contains either one of the (S)-isomer compoundrepresented by General Formula (3S):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (3)) or the (R)-isomer compound represented by General Formula(3R):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (3)), or may be a racemic compound having a weight ratio of 1:1.The compound is preferably an (S)-isomer compound since, by the steps(ii), (iii), and (iv) described later, the (S)-isomer can be convertedto an α-substituted-D-cysteine or a salt thereof, which is useful as anintermediate for pharmaceuticals and the like, among the α-substitutedcysteines represented by General Formula (1) and salts thereof.

Examples of the compound represented by the General Formula (3S) includethe following compounds.

Among the compounds described above, the following compounds arepreferred as the compound represented by General Formula (3S).

Examples of the compound represented by the General Formula (3R) includethe following compounds.

Among the compounds described above, the following compounds arepreferred as the compound represented by General Formula (3R).

In the step (i), a base or an acid, or the enzyme and/or the like of thepresent invention is/are allowed to act on a compound represented by theGeneral Formula (2). The respective cases are described below in detail.

(Cases of Reaction with a Base)

In cases where a base is allowed to act for the hydrolysis, examples ofthe base include alkali metal hydroxides such as sodium hydroxide,lithium hydroxide, and potassium hydroxide; and alkali metal carbonatessuch as lithium carbonate, sodium carbonate, and potassium carbonate.These bases may be used individually, or as a mixture of two or more ofthese.

The amount of the base used may be appropriately set within the rangeof, for example, 0.7 molar equivalent to 10 molar equivalents withrespect to the amount of the compound represented by General Formula(2). From the viewpoint of the yield and the cost, the amount ispreferably 0.9 molar equivalent to 2 molar equivalents. From theviewpoint of prevention of further hydrolysis of the ester group in thecompound represented by General Formula (3), the amount is morepreferably 0.9 molar equivalent to 1.2 molar equivalents.

In cases where a base is allowed to act, the reaction temperature may beappropriately selected within the range of, for example, −50° C. to 80°C. For enabling simple control of the temperature to suppress sidereactions, the reaction temperature is preferably within the range of 0°C. to 40° C.

In cases where a base is allowed to act, the reaction time may beappropriately selected within the range of, for example, 0.5 hour to 50hours. The reaction time is preferably within the range of 2 hours to 10hours.

In cases where a base is allowed to act, the reaction solvent is usuallywater alone, or a mixed solvent of water and an organic solvent.Examples of the organic solvent include alcohols such as glycerol,ethylene glycol, methanol, ethanol, 1-propanol, 2-propanol, andt-butanol; ketones such as acetone, 2-butanone, and methylisobutylketone; ethers such as diethyl ether, di-n-propyl ether, diisopropylether, di-n-butyl ether, methyl isopropyl ether, methyl-t-butyl ether,ethyl-t-butyl ether, cyclopentyl methyl ether, tetrahydrofuran, dioxane,and 1,2-dimethoxyethane; aliphatic hydrocarbons such as n-hexane andn-heptane; esters such as ethyl acetate, isopropyl acetate, and butylacetate; aromatic hydrocarbons such as benzene, toluene, and xylene;nitriles such as acetonitrile and propionitrile; halogenatedhydrocarbons such as dichloromethane, chloroform, carbon tetrachloride,and 1,2-dichloroethane; dimethylsulfoxide; and dimethylformamide.

In particular, in order to increase the reaction rate, a uniformreaction mixture is preferably provided using water, in which alkalimetal hydroxides and alkali metal carbonates are highly soluble, and awater-soluble solvent in which the compound represented by GeneralFormula (2) is highly soluble and which is highly miscible with water.Examples of the water-soluble solvent include alcohols such as glycerol,ethylene glycol, methanol, ethanol, 1-propanol, 2-propanol, andt-butanol; ketones such as acetone and 2-butanone; ethers such astetrahydrofuran, dioxane, and 1,2-dimethoxyethane; nitriles such asacetonitrile and propionitrile; dimethylsulfoxide; anddimethylformamide. In particular, from the viewpoint of ease of removalof the solvent by distillation, methanol, ethanol, acetone, andtetrahydrofuran are more preferred because of their low boiling points.

In the step (i) of the present invention, the reaction is morepreferably carried out with a base than with an acid, since a base cansuppress the side reaction of hydrolyzing both, rather than one, of theester groups contained in General Formula (2), and therefore enablesproduction of the compound represented by General Formula (3) with highyield.

(Cases of Reaction with an Acid)

In cases where an acid is allowed to act on the compound represented byGeneral Formula (2) to perform hydrolysis, examples of the acid includeinorganic acids such as hydrochloric acid, sulfuric acid, phosphoricacid, hydrogen bromide, hydrogen fluoride, and hydrogen iodide; andorganic acids such as trifluoroacetic acid, trifluoromethanesulfonicacid, methanesulfonic acid, and p-toluenesulfonic acid(p-toluenesulfonic acid is preferably monohydrate; the same applieshereinafter). These acids may be used individually, or as a mixture oftwo or more thereof.

The amount of the acid used may be appropriately set within the rangeof, for example, 0.1 molar equivalent to 20 molar equivalents withrespect to the amount of the compound represented by General Formula(2). From the viewpoint of the yield and the cost, the amount ispreferably 1.0 molar equivalent to 10 molar equivalents.

In cases where an acid is allowed to act, the reaction temperature maybe appropriately selected within the range of, for example, 0° C. to200° C. For reducing the reaction time and enabling simple control ofthe temperature, the reaction temperature is preferably within the rangeof 30° C. to 100° C.

In cases where an acid is allowed to act, the reaction time may beappropriately selected within the range of, for example, 0.5 hour to 50hours. The reaction time is preferably within the range of 2 hours to 20hours.

In cases where a base is allowed to act, the reaction solvent is usuallywater alone, or a mixed solvent of water and an organic solvent.Examples of the organic solvent include ketones such as acetone,2-butanone, and methylisobutyl ketone; ethers such as diethyl ether,di-n-propyl ether, diisopropyl ether, di-n-butyl ether, methyl isopropylether, methyl-t-butyl ether, ethyl-t-butyl ether, cyclopentyl methylether, tetrahydrofuran, dioxane, and 1,2-dimethoxyethane; aliphatichydrocarbons such as n-hexane and n-heptane; esters such as ethylacetate, isopropyl acetate, and butyl acetate; aromatic hydrocarbonssuch as benzene, toluene, and xylene; nitriles such as acetonitrile andpropionitrile; halogenated hydrocarbons such as dichloromethane,chloroform, carbon tetrachloride, and 1,2-dichloroethane;dimethylsulfoxide; and dimethylformamide.

From the viewpoint of stability against acids, preferred examples of theorganic solvent include aliphatic hydrocarbons such as n-hexane andn-heptane; esters such as ethyl acetate, isopropyl acetate, and butylacetate; aromatic hydrocarbons such as benzene, toluene, and xylene;nitriles such as acetonitrile and propionitrile; halogenatedhydrocarbons such as dichloromethane, chloroform, carbon tetrachloride,and 1,2-dichloroethane; dimethylsulfoxide; and dimethylformamide.

(Cases of Reaction with the Enzyme and/or the Like of the PresentInvention)

The compound represented by General Formula (3) can also be obtained byallowing the enzyme and/or the like of the present invention, that is,an enzyme having an activity to hydrolyze an ester group, a cell havingan ability to produce the enzyme, a processed product of the cell,and/or a culture liquid containing the enzyme obtained by culturing thecell, to act on a compound represented by General Formula (2).

In particular, in cases where a chiral compound which selectivelycontains either one of General Formulae (3S) and (3R) is to be obtainedas General Formula (3), the enzyme and/or the like of the presentinvention is preferably allowed to act rather than the above-describedacid or base, from the viewpoint of stereoselectivity.

The enzyme having an activity to hydrolyze an ester group that may beused in the production method of the present invention is not limited aslong as the enzyme has the activity, and examples of the enzyme includeenzymes comprising the amino acid sequence of (A) SEQ ID NO: 4, 6, 8,10, 12, 14, 16, or 18. These are carboxyesterase NP derived fromBacillus subtilis.

In the present invention, their homologues having the enzyme activitymay also be used. Examples of the homologues include (B) and (C)described below.

(B) A protein having an identity of not less than 35% to the amino acidsequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18, and having anactivity to hydrolyze a compound represented by the General Formula (2)for conversion into a compound represented by General Formula (3S):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (3)), which is a compound represented by the General Formula (3)and having an (S)-configuration.

(C) A protein comprising the amino acid sequence of SEQ ID NO: 4, 6, 8,10, 12, 14, 16, or 18 in which one or several amino acids are deleted,substituted, and/or added, and having an activity to hydrolyze acompound represented by the General Formula (2) for conversion into acompound represented by General Formula (3S), which is a compoundrepresented by the General Formula (3) and having an (S)-configuration.

The protein (B) herein may have an identity of not less than 35%,preferably not less than 50%, more preferably not less than 80%,especially preferably not less than 95%, most preferably not less than98% to the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or18, as long as the protein has an activity to hydrolyze an ester group.For example, the identity search for the protein can be carried outusing a program such as FASTA or BLAST (Basic Local Alignment SearchTool) against GenBank or DNA Databank of JAPAN (DDBJ).

Examples of the protein (C) include proteins comprising the amino acidsequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18 in which one orseveral amino acids are deleted, substituted, and/or added, as long asthe activity to hydrolyze an ester group is not deteriorated. Morespecifically, the term “several” herein means not more than 20,preferably not more than 10, more preferably not more than 5.

The enzyme having an activity to hydrolyze an ester group used in thepresent invention can be obtained by isolating DNA encoding the enzymefrom an arbitrary microorganism having the enzyme activity using a probeprepared based on the nucleotide sequence of a gene encoding a part of,or the whole enzyme, and then transforming a host organism such as E.coli with the DNA, followed by allowing the host organism to express theenzyme.

The enzyme having an activity to hydrolyze an ester group used in thepresent invention can also be obtained by purification from cells of amicroorganism having the enzyme activity, for example, a Bacillusbacterium.

Examples of the Bacillus bacterium include Bacillus licheniformis,Bacillus subtilis, and Bacillus stearothermophilus. Their strains areavailable from National Institute of Technology and Evaluation.Alternatively, the strains can be obtained from ATCC (American TypeCulture Collection) in cases where they are described in the onlinecatalog (http://www.atcc.org/) by ATCC.

In the production method of the present invention, the enzyme may beallowed to act on a compound represented by General Formula (2).Alternatively, instead of the enzyme, or in addition to the enzyme, acell having an ability to produce the enzyme, a processed product of thecell, and/or a culture liquid containing the enzyme obtained byculturing the cell, may be allowed to act on the compound. The cellhaving an ability to produce the enzyme is preferably a cell transformedwith DNA encoding the enzyme. The cell may be a microorganism (hostorganism), cultured cell, or the like, and may be either a living cellor a dead cell. For example, a resting microorganism may also befavorably used.

Examples of the processed product of the cell include treated productsof the cell, such as the cell treated with an organic solvent, forexample, acetone, dimethylsulfoxide (DMSO), or toluene, the cell treatedwith a surfactant, the cell treated by freeze drying, and the cellmechanically or enzymatically homogenized; crude purified products orpurified products of an enzyme fraction extracted from the cell; andcarriers such as polyacrylamide gel and carrageenan gel on which theseproducts are immobilized.

As the culture liquid obtained by culturing the cell, a culture liquidobtained by culturing the cell, that is, a culture liquid containing theenzyme, may be directly used. Alternatively, for example, a suspensionof the cell in a liquid medium may be used, or, in cases where the cellis of a secretory expression type, a supernatant obtained after removalof the cell by centrifugation or the like, or a concentrated productthereof, may be used.

Examples of the DNA encoding the enzyme to be used for thetransformation or the like herein include DNAs encoding a proteincomprising the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14,16, or 18. Alternatively, the DNA may be a DNA encoding a proteincomprising an amino acid sequence with a homology of not less than 35%,preferably not less than 50%, more preferably not less than 80%,especially preferably not less than 95%, most preferably not less than98% to the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or18, and having an enzyme activity to convert a compound represented byGeneral Formula (2) to a compound represented by the General Formula(3).

Examples of the DNA encoding an enzyme comprising the amino acidsequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18 include DNAscomprising the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15,or 17, respectively. The DNA may be a homologue of the nucleotidesequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17, as long as the DNAencodes a protein having an activity to hydrolyze an ester group. Thatis, examples of the DNA encoding the enzyme include the following.

(D) A DNA comprising the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9,11, 13, 15, or 17.

(E) A DNA comprising the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9,11, 13, 15, or 17 in which one or several bases are substituted,deleted, and/or added, and encoding a polypeptide having an enzymeactivity to convert a compound represented by the General Formula (2) toa compound represented by the General Formula (3).

(F) A DNA comprising the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9,11, 13, 15, or 17; or a DNA comprising a nucleotide sequence whichhybridizes with the complementary strand thereof under stringentconditions, and encoding a polypeptide having an enzyme activity toconvert a compound represented by the General Formula (2) to a compoundrepresented by the General Formula (3).

The nucleotide sequences of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, and 17are cDNA sequences.

More specifically, in the DNA of (E), the term “several” means not morethan 60, preferably not more than 30, more preferably not more than 10,most preferably not more than 5.

In the DNA of (F), the “DNA which hybridizes under stringent conditions”means a DNA obtained by carrying out colony hybridization, plaquehybridization, Southern blot hybridization, or the like using a probeDNA under stringent conditions. Examples of the “stringent conditions”include conditions for colony hybridization and plaque hybridizationwherein hybridization is carried out in the presence of 0.7 mol/L to 1.0mol/L sodium chloride at 65° C. using a filter on which colony-derivedor plaque-derived DNAs or fragments of the DNAs are immobilized, and theresulting filter is then washed using 0.1 to 2×SSC solution (compositionof 1×SSC: 150 mmol/L sodium chloride and 15 mmol/L sodium citrate) at65° C.

The DNA encoding an enzyme having an activity to hydrolyze an estergroup can be isolated by, for example, the following method. First, anenzyme having an activity to hydrolyze an ester group is purified frommicroorganism cells or the like by the above-described method or thelike, and the amino acid sequence at the N-terminus of the enzyme isanalyzed. The analysis of the amino acid sequence at the N-terminus iscarried out by cleaving the purified protein with enzymes such as lysylendopeptidase, V8 protease, and/or the like, purifying the resultingpeptide fragments by reversed-phase liquid chromatography and/or thelike, and then determining a plurality of amino acid sequences by aminoacid sequence analysis using a protein sequencer. By performing PCRusing primers designed based on the amino acid sequences determined, andchromosomal DNA or a cDNA library of a microorganism strain thatproduces the enzyme as a template, a part of DNA (DNA fragment) encodingthe enzyme can be obtained. A restriction enzyme digest of chromosomalDNA of a microorganism strain that produces the enzyme is introducedinto a phage, plasmid, or the like, and E. coli is transformed with theresulting phage, plasmid, or the like to obtain a library or a cDNAlibrary. By carrying out colony hybridization, plaque hybridization, orthe like against the resulting library or cDNA library using the DNAfragment as a probe, a DNA encoding the enzyme having an activity tohydrolyze an ester group can be obtained.

It is also possible to obtain the DNA encoding the enzyme by analyzingthe nucleotide sequence of the DNA fragment obtained by the PCR,designing, based on the sequence obtained, a PCR primer for extension ofthe sequence to the outside of the region whose sequence was determined,digesting chromosomal DNA of a microorganism strain which produces theenzyme with an appropriate restriction enzyme, and then performinginverse PCR (Genetics vol. 120, p 621-623 (1988)) using DNA cyclized byself-cyclization as a template. The DNA comprising the sequence of SEQID NO: 3, 5, 7, 9, 11, 13, 15, or 17 herein can be obtained by PCR usingprimers designed based on the nucleotide sequence of SEQ ID NO: 3, 5, 7,9, 11, 13, 15, or 17, respectively.

The DNA encoding the enzyme can also be obtained by chemical synthesisof DNA having the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13,15, or 17.

Those skilled in the art can obtain the DNA encoding the enzyme havingan activity to hydrolyze an ester group which can be used in theproduction method of the present invention, by introducing, ifnecessary, a substitution, deletion, insertion, and/or additionmutation(s) to the DNA of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17 bysite-directed mutagenesis (Adv. Biochem. Eng. vol. 43, p 75-102 (1990);Yeast vol., Molecular Cloning 2nd Edt., Cold Spring Harbor LaboratoryPress (1989); PCR: A Practical Approach, IRL Press, p200 (1991)) or thelike.

Alternatively, based on the whole or part of the amino acid sequence ofSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18, or the whole or part of thenucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17,homology search may be carried out against a database such as GenBank orDDBJ to obtain nucleotide sequence information on a DNA homolog encodingan enzyme having an activity to hydrolyze an ester group. Those skilledin the art can obtain DNA encoding the enzyme by PCR or the like from astrain based on the nucleotide sequence information.

The DNA encoding an enzyme having an activity to hydrolyze an estergroup can also be obtained by carrying out colony hybridization, plaquehybridization, Southern blot hybridization, or the like using a DNAhaving the whole or part of the nucleotide sequence of SEQ ID NO: 3, 5,7, 9, 11, 13, 15, or 17 as a probe under stringent conditions againstDNA prepared from an arbitrary microorganism having the enzyme activity,and obtaining a hybridizing DNA. The “part” herein means a DNA having alength sufficient for use as a probe. More specifically, the length ofthe DNA is not less than 15 bp, preferably not less than 50 bp, morepreferably not less than 100 bp.

Each hybridization can be carried out according to the method describedin Chapter 1 of Molecular Cloning 3rd Edt. (Cold Spring HarborLaboratory Press, 2001)).

By inserting the thus isolated DNA encoding an enzyme having an activityto hydrolyze an ester group into a known expression vector in a mannerwhich allows expression of the enzyme, an expression vector for theenzyme having an activity to hydrolyze an ester group can be provided.By culturing cells transformed with this expression vector, the enzymecan be obtained from the cells. Transformed cells can also be obtainedby incorporating the DNA encoding an enzyme having an activity tohydrolyze an ester group into chromosomal DNA of known host cells in amanner which allows expression of the enzyme.

More specifically, the preparation of the transformed cells needs to becarried out by incorporating the DNA encoding an enzyme having anactivity to hydrolyze an ester group into a plasmid vector or a phagevector capable of being stably present in a microorganism, andintroducing the constructed expression vector into the microorganism, orby directly introducing the DNA encoding the enzyme into the genome of ahost organism, and allowing transcription and translation of the geneticinformation.

In cases where the DNA encoding the enzyme does not contain a promoterwhich allows expression in the host microorganism, an appropriatepromoter needs to be incorporated in the 5′-upstream in the DNA strandwhere the enzyme is encoded. In addition, a terminator is preferablyincorporated in the 3′-downstream. The promoter and the terminator arenot limited as long as these are a promoter and a terminator which areknown to function in the microorganism used as the host. Vectors,promoters, and terminators which can be used for the variousmicroorganisms are described in detail in, for example, “FundamentalMicrobiology, Vol. 8, Genetic Engineering, Kyoritsu Shuppan Co., Ltd.”;and, especially for cases of yeasts, Adv. Biochem. Eng. vol. 43, p75-102 (1990); and Yeast vol. 8, p 423-488 (1992).

The host organism to be transformed for expression of the enzyme havingan activity to hydrolyze an ester group of the present invention is notlimited as long as the host organism does not adversely affect thereaction. Specific examples of the host organism include the followingmicroorganisms.

Bacteria belonging to the genera Escherichia, Bacillus, Pseudomonas,Serratia, Brevibacterium, Corynebacterium, Streptococcus, Lactobacillus,and the like whose host-vector systems are established.

Actinomycetes belonging to the genera Rhodococcus, Streptomyces, and thelike whose host-vector systems are established.

Yeasts belonging to the genera Saccharomyces, Kluyveromyces,Schizosaccharomyces, Zygosaccharomyces, Yarrowia, Trichosporon,Rhodosporidium, Hansenula, Pichia, Candida, and the like whosehost-vector systems are established.

Molds belonging to the genera Neurospora, Aspergillus, Cephalosporium,Trichoderma, and the like whose host-vector systems are established.

Among these, the genera Escherichia, Bacillus, Brevibacterium, andCorynebacterium are preferred, and the genera Escherichia andCorynebacterium are especially preferred as the host microorganism.

The procedure for the preparation of the transformed cells, theconstruction of the recombinant vector suitable for the host organism,and the method for culturing the host can be carried out according totechniques conventionally used in the fields of molecular biology,bioengineering, and genetic engineering (see, for example, MolecularCloning 3rd Edt. (Cold Spring Harbor Laboratory Press, 2001)).

Various host-vector systems have been established in plants and animals,in addition to microorganisms. In particular, systems that allowexpression of a large amount of a heterogeneous protein in cells of ananimal such as an insect (e.g., silkworm) (Nature vol. 315, p 592-594(1985)), or cells of a plant such as rapeseed, maize, or potato; andsystems using a cell-free protein synthesis system based on a cell-freeextract from E. coli, wheat germ, or the like; have been established,and may be preferably used.

Specific examples of the enzyme having an activity to hydrolyze an estergroup include Bacillus licheniformis-derived protease (manufactured bySigma-Aldrich), Bacillus subtilis-derived carboxyesterase NP (SEQ ID NO:4, 6, 8, 10, 12, 14, 16, or 18), Bacillus stearothermophilus-derivedesterase BS1 (manufactured by Julich Fine Chemicals), Bacillusstearothermophilus-derived esterase BS3 (manufactured by Julich FineChemicals), and pig liver-derived esterase (manufactured bySigma-Aldrich), and these may be appropriately selected depending on thepurpose.

Among these, in cases where a chiral compound selectively containing an(S)-isomer compound represented by General Formula (3S) is to beobtained, Bacillus licheniformis-derived protease (manufactured bySigma-Aldrich) and Bacillus subtilis-derived carboxyesterase NP arepreferred as the enzyme.

The optical purity of the compound represented by General Formula (3S)obtained in this case is generally not less than 90.0% e.e., but, sincehigh optical purity is generally required in cases where the compound isproduced as a pharmaceutical or an intermediate therefor, the opticalpurity is preferably not less than 95.0% e.e., more preferably not lessthan 99.0% e.e., especially preferably not less than 99.5% e.e., mostpreferably not less than 99.8% e.e.

In cases where a chiral compound selectively containing an (R)-isomercompound represented by General Formula (3R) is to be obtained, Bacillusstearothermophilus-derived esterase BS1 (manufactured by Julich FineChemicals), Bacillus stearothermophilus-derived esterase BS3(manufactured by Julich Fine Chemicals), or pig liver-derived esterase(manufactured by Sigma-Aldrich) is preferably used as the enzyme.Bacillus stearothermophilus-derived esterase BS1 (manufactured by JulichFine Chemicals), Bacillus stearothermophilus-derived esterase BS3(manufactured by Julich Fine Chemicals), and the like are preferred.

The optical purity of the compound represented by General Formula (3R)obtained in this case is generally not less than 90.0% e.e., but, sincehigh optical purity is generally required in cases where the compound isproduced as a pharmaceutical or an intermediate therefor, the opticalpurity is preferably not less than 95.0% e.e., more preferably not lessthan 99.0% e.e., especially preferably not less than 99.5% e.e., mostpreferably not less than 99.8% e.e.

In cases where the hydrolysis is carried out by action of an enzyme, acell having an ability to produce the enzyme, a processed product of thecell, and/or a culture liquid containing the enzyme obtained byculturing the cell, the hydrolysis is usually carried out in an aqueoussolvent. However, the hydrolysis may also be carried out in the presenceof at least one organic solvent.

In cases where an organic solvent is used, examples of the organicsolvent which may be used include alcohols such as glycerol, ethyleneglycol, methanol, ethanol, 1-propanol, and 2-propanol; ketones such asacetone, 2-butanone, and methylisobutyl ketone; ethers such as diethylether, di-n-propyl ether, diisopropyl ether, di-n-butyl ether, methylisopropyl ether, methyl-tert-butyl ether, ethyl-tert-butyl ether,cyclopentyl methyl ether, tetrahydrofuran, dioxane, and1,2-dimethoxyethane; aliphatic hydrocarbons such as n-pentane, n-hexane,and n-heptane; esters such as ethyl acetate, isopropyl acetate, andbutyl acetate; aromatic hydrocarbons such as benzene, toluene, andxylene; nitriles such as acetonitrile and propionitrile; halogenatedhydrocarbons such as dichloromethane, chloroform, carbon tetrachloride,and 1,2-dichloroethane; dimethylsulfoxide; and dimethylformamide.Preferably, from the viewpoint of organic solvent tolerance of theenzyme, use of an organic solvent is avoided, and only water is used.

The reaction temperature may be selected within the range of, forexample, 0° C. to 80° C. From the viewpoint of thermal stability of theenzyme, the reaction temperature is preferably 20° C. to 50° C.

The reaction time may be appropriately selected within the range of, forexample, 1 hour to 120 hours. The reaction time is preferably 2 hours to40 hours since, in cases where the reaction time is long, the yieldtends to decrease due to hydrolysis of the ester group in the compoundrepresented by General Formula (3).

A pH suitable for the reaction may be appropriately selected dependingon the enzyme used and the like. For example, the pH is 2.0 to 12.0. ThepH is preferably 6.0 to 11.0, more preferably 7.0 to 9.0. Such a pH isselected for prevention of hydrolysis in which the enzyme is notinvolved. In the hydrolysis in which the enzyme is not involved, theester group in the compound represented by General Formula (3) mayundergo hydrolysis after the reaction of interest, leading to a decreasein the yield; or a compound represented by General Formula (3R) may beunexpectedly produced in the reaction to obtain a compound representedby General Formula (3S), or a compound represented by General Formula(3S) may be unexpectedly produced in the reaction to obtain a compoundrepresented by General Formula (3R), leading to a decrease in thepurity. Thus, the reaction is preferably carried out at a pH at whichthese phenomena can be suppressed.

Also in cases where the enzyme and/or the like of the present inventionis allowed to act, an acid or a base may be added in order to adjust thepH within a preferred range.

(Post-Treatment)

After the reaction, if necessary, post-treatment may be carried out by,for example, inactivation of the microorganism, centrifugation,coagulant treatment, filtration, extraction, concentration, and/orpurification on a column. In cases where extraction is carried out, anacid may be added to the reaction solution to release the compound ofGeneral Formula (3) as a carboxylic acid, and extraction using awater-insoluble organic solvent may be performed. In cases where thepurity of the compound represented by General Formula (3) issufficiently high even without purification, it is preferred to carryout only filtration, extraction, and concentration without carrying outpurification, from the viewpoint of simplicity. More preferably, incases where extraction is carried out, the extraction is carried outwith the reaction solvent used in the subsequent step, and onlyfiltration and the extraction are carried out.

Examples of the water-insoluble organic solvent used in the extractioninclude ketones such as 2-butanone, methylisobutyl ketone, andcyclohexanone; aromatic hydrocarbons such as benzene, toluene, xylene,chlorobenzene, and trifluoromethylbenzene; aliphatic hydrocarbons suchas n-pentane, n-hexane, n-heptane, and cyclohexane; esters such asmethyl acetate, ethyl acetate, isopropyl acetate, and butyl acetate;ethers such as diethyl ether, di-n-propyl ether, diisopropyl ether,di-n-butyl ether, methyl-tert-butyl ether, ethyl-tert-butyl ether, andcyclopentyl methyl ether; and halogenated hydrocarbons such asdichloromethane, chloroform, carbon tetrachloride, and1,2-dichloroethane. Two or more of these may be used as a mixed solvent.

Among these, from the viewpoint of extraction efficiency, aromatichydrocarbons and esters are preferred. Use of benzene, toluene, methylacetate, ethyl acetate, isopropyl acetate, butyl acetate, or a mixedsolvent of two or more of these is more preferred. Use of toluene isstill more preferred since toluene has high tolerance to acidicconditions, and can be reused.

<Step (ii)>

The step (ii) is described below.

The step (ii) is a step of allowing a condensing agent or an acidhalogenating agent to act on a compound represented by General Formula(3):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² represents aC₁-C₄ alkyl group), to obtain a compound represented by General Formula(4):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (3); X represents —OP(O)(OPh)₂, —OP(O)(OEt)₂, —OC(O)OR³, or ahalogen atom; and R³ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted).

The R¹ and the R² in the compounds represented by the General Formulae(3) and (4) are the same as those described in the step (i).

The compound represented by the General Formula (4) may be either aracemic compound or a chiral compound. Since high optical purity isgenerally required in cases where the compound is produced as apharmaceutical or an intermediate therefor, the compound is preferably achiral compound having high optical purity. The optical purity ispreferably not less than 95.0% e.e., more preferably not less than 99.0%e.e., especially preferably not less than 99.5% e.e., most preferablynot less than 99.8% e.e.

Examples of X in General Formula (4) include a —OP(O)(OPh)₂ group,—OP(O)(OEt)₂ group, —OC(O)OR³ group (wherein R³ represents a C₁-C₁₀alkyl group which is optionally substituted, a C₇-C₂₀ aralkyl groupwhich is optionally substituted, or a C₆-C₁₂ aryl group which isoptionally substituted), and a halogen atom. Among these, compoundsrepresented by General Formula (4-1):

(wherein R¹, R², and R³ have the same meanings as R¹, R², and R³ in theGeneral Formula (4)), wherein X represents —OC(O)OR³, are preferredsince they are stable against water and can be easily handled in thelater-described step (iii).

Examples of the alkyl group of the “C₁-C₁₀ alkyl group which isoptionally substituted” as R³ include C₁-C₁₀ linear, branched, or cyclicalkyl groups such as methyl, ethyl, isopropyl, n-propyl, n-butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl,neopentyl, n-hexyl, cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl. Examples of the substituent optionally contained in thealkyl group include C₁-C₆ alkoxy groups such as methoxy and ethoxy;halogen atoms such as a fluorine atom, chlorine atom, bromine atom, andiodine atom; and nitro. The number of substituents is not limited, and,in cases where there are two or more substituents in the alkyl group,the substituents may be of the same type or different types.

Examples of the aralkyl group of the “C₇-C₂₀ aralkyl group which isoptionally substituted” as R³ include benzyl, 2-phenylethyl,1-phenylethyl, 1-phenylpropyl, 2-phenylpropyl, and 3-phenylpropyl.Examples of the substituent optionally contained in the aralkyl groupinclude C₁-C₆ alkyl groups such as methyl, ethyl, and isopropyl; C₁-C₆alkoxy groups such as methoxy and ethoxy; halogen atoms such as afluorine atom, chlorine atom, bromine atom, and iodine atom; and nitro.The number of substituents is not limited, and, in cases where there aretwo or more substituents in the aralkyl group, the substituents may beof the same type or different types.

Examples of the aryl group of the “C₆-C₁₂ aryl group which is optionallysubstituted” as R³ include phenyl, 1-naphthyl, and 2-naphthyl. Examplesof the substituent optionally contained in the aryl group include C₁-C₆alkyl groups such as methyl, ethyl, and isopropyl; C₁-C₆ alkoxy groupssuch as methoxy and ethoxy; halogen atoms such as a fluorine atom,chlorine atom, bromine atom, and iodine atom; and nitro. The number ofsubstituents is not limited, and, in cases where there are two or moresubstituents in the aryl group, the substituents may be of the same typeor different types.

For each type of X in the General Formula (4), preferred condensingagents or acid halogenating agents, and preferred structures of X aredescribed below.

In cases where a compound represented by the General Formula (4) inwhich X is —OP(O)(OPh)₂, that is, a compound represented by the GeneralFormula (4-1), is to be obtained, diphenylphosphoryl azide may be usedas a condensing agent.

In cases where a compound represented by the General Formula (4) inwhich X is —OP(O)(OEt)₂ is to be obtained, diethylphosphoryl azide maybe used as a condensing agent.

In cases where a compound represented by the General Formula (4) inwhich X is —OC(O)OR³ is to be obtained, a chloroformic ester representedas ClC(O)OR³ may be used as a condensing agent or an acid halogenatingagent. Preferred examples of easily available chloroformic estersinclude C₁-C₆ linear or branched chloroformic esters such as methylchloroformate, ethyl chloroformate, n-propyl chloroformate, isopropylchloroformate, n-butyl chloroformate, isobutyl chloroformate, andtert-butyl chloroformate; phenyl chloroformate; and benzylchloroformate.

In the cases where X is —OC(O)OR³, R³ is preferably a C₁-C₆ linear orbranched alkyl group such as methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, or tert-butyl; phenyl; or benzyl; from the viewpointof availability as the chloroformic ester.

The structure of R³ also influences the later-described step (iii). As amixed acid anhydride, the compound represented by the General Formula(4-1) has both a carbonyl group derived from a substrate malonic acidderivative and a carbonyl group derived from a condensing agent. Thereaction in the step (iii) begins by nucleophilic attack by an azideanion. In cases where the nucleophilic attack does not occur selectivelyto the carbonyl group of interest derived from the malonic acidderivative, the yield of the compound represented by General Formula(5):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (4)) tends to decrease. Normally, considering the electrondensity of the two carbonyl groups, the nucleophilic attack of interestoccurs. However, since the compound represented by General Formula (4-1)is a disubstituted malonic acid derivative, the extent of sterichindrance in the vicinity of the carbonyl group derived from the malonicacid derivative is large. Therefore, the nucleophilic attack of interestmay be unlikely to occur selectively especially in cases where thestructure of R³ in the compound represented by General Formula (4-1) issmall.

Thus, R³ is more preferably ethyl, n-propyl, isopropyl, n-butyl,isobutyl, tert-butyl, phenyl, or benzyl. As the chloroformic estercorresponding thereto, ethyl chloroformate, n-propyl chloroformate,isopropyl chloroformate, n-butyl chloroformate, isobutyl chloroformate,tert-butyl chloroformate, phenyl chloroformate, or benzyl chloroformateis preferred. In the later-described step (iii), the alcohol representedby R³OH is produced as a by-product, and the by-produced alcohol needsto be removed by washing with water or concentration. Considering easeof the removal of the alcohol, R³ is still more preferably ethyl orisopropyl. The chloroformic esters corresponding to these are ethylchloroformate and isopropyl chloroformate.

Thus, specific examples of the compound represented by the GeneralFormula (4-1) include the following compounds.

Among the compounds mentioned above, the following compounds arepreferred as the compound represented by the General Formula (4-1).

In cases where a compound represented by the General Formula (4) inwhich X is a halogen atom is to be obtained, an acid halogenating agentmay be used in the step (ii).

Examples of the halogen atom herein include a chlorine atom, bromineatom, and iodine atom. From the viewpoint of stability of the compound,a chloride atom is preferred.

Examples of the acid halogenating agent include thionyl chloride,sulfuryl chloride, thionyl bromide, phosphorous trichloride, phosphorouspentachloride, phosphorous oxytrichloride, phosphorus tribromide,phosphorus pentabromide, phosphorous oxytribromide, oxalyl chloride,oxalyl bromide, phosgene, triphosgene, and cyanuric chloride. Inparticular, since X is preferably a chlorine atom as described above,thionyl chloride, sulfuryl chloride, phosphorous trichloride,phosphorous pentachloride, phosphorous oxytrichloride, oxalyl chloride,phosgene, triphosgene, and cyanuric chloride are preferred. Thionylchloride, sulfuryl chloride, and oxalyl chloride are more preferredbecause of their low toxicity.

The conditions for the reaction with the condensing agent or the acidhalogenating agent vary depending on, for example, the type of X in theGeneral Formula (4) and the structure of R³, and may therefore beappropriately set. For example, the reaction may be carried out underthe following reaction conditions.

Normally, the amount of the condensing agent or the acid halogenatingagent may be appropriately set within the range of 0.7 molar equivalentto 10 molar equivalents with respect to the amount of the compoundrepresented by General Formula (3). In order to allow completedisappearance of the material and to increase the yield, the amount ispreferably 1.0 molar equivalent to 5.0 molar equivalents. From theviewpoint of reducing the purification load, the amount is morepreferably 1.0 molar equivalent to 1.5 molar equivalents.

A base may be used in the reaction by the condensing agent or the acidhalogenating agent. The base is not limited as long as the base does nothave nucleophilicity, and tertiary amines and pyridines are preferred asthe base.

Examples of the tertiary amines include trimethylamine, triethylamine,triisopropylamine, tripropylamine, triisobutylamine,N,N-dimethylethylamine, N,N-dimethylisopropylamine,N-butyldimethylamine, N,N-diisopropylethylamine,N,N-dimethylcyclohexylamine, and N-methylmorpholine.

Examples of the pyridines include pyridine, 2-chloropyridine,3-chloropyridine, 2-methylpyridine, 3-methylpyridine, 2,3-lutidine,2,4-lutidine, 2,5-lutidine, 2,6-lutidine, 3,4-lutidine, and3,5-lutidine.

Among the bases described above, the tertiary amines are preferred. Thisis because the salt produced by neutralization of the compoundrepresented by General Formula (4) by the base is easily soluble inorganic solvents, and the reaction can be allowed to proceed smoothly asa result. Triethylamine is more preferred among the tertiary aminessince it is inexpensive.

The amount of the base used may be appropriately set within the rangeof, for example, 0.7 molar equivalent to 10 molar equivalents withrespect to the amount of the compound represented by General Formula(3). From the viewpoint of allowing complete disappearance of thematerial and increasing the yield, the amount is preferably 1.0 molarequivalent to 5.0 molar equivalents. From the viewpoint of reduction ofthe purification load, the amount is more preferably 1.0 molarequivalent to 1.5 molar equivalents. From the viewpoint of suppressionof production of by-products, the base is still more preferably used inthe same amount as that of the condensing agent or the acid halogenatingagent in terms of molar equivalence.

Examples of the reaction solvent used for the reaction by the condensingagent or the acid halogenating agent include ketones such as acetone,2-butanone, methylisobutyl ketone, and cyclohexanone; aromatichydrocarbons such as benzene, toluene, xylene, chlorobenzene, andtrifluoromethylbenzene; aliphatic hydrocarbons such as n-pentane,n-hexane, n-heptane, and cyclohexane; esters such as methyl acetate,ethyl acetate, isopropyl acetate, and butyl acetate; ethers such asdiethyl ether, di-n-propyl ether, diisopropyl ether, di-n-butyl ether,methyl isopropyl ether, methyl-tert-butyl ether, ethyl-tert-butyl ether,cyclopentyl methyl ether, tetrahydrofuran, dioxane, and1,2-dimethoxyethane; amides such as N,N-dimethylformamide andN,N-dimethylacetamide; nitriles such as acetonitrile and propionitrile;halogenated hydrocarbons such as dichloromethane, chloroform, carbontetrachloride, and 1,2-dichloroethane; and dimethylsulfoxide.

Among these, acetone, toluene, xylene, ethyl acetate, isopropyl acetate,tetrahydrofuran, dioxane, N,N-dimethylformamide, N,N-dimethylacetamide,acetonitrile, and propionitrile are preferred since the salt produced byneutralization of the compound represented by the General Formula (4) bythe base is highly soluble in these solvents, and high reactionefficiency can be achieved. Acetone, toluene, xylene, ethyl acetate,isopropyl acetate, and tetrahydrofuran are more preferred from theviewpoints of their inexpensiveness and availability, and ofsimplification of the process since these do not adversely affect thesubsequent step (iii) even without post-treatment. In cases where apolar solvent such as water or an alcohol is used, reaction tends tooccur with the condensing agent or the acid halogenating agent, or thecompound represented by the General Formula (4), leading to a decreasein the yield.

The amount of the reaction solvent used may be appropriately set withinthe range of, for example, 0 volume to 100 volumes with respect to theamount of the compound represented by the General Formula (3). Theamount of the reaction solvent is preferably 1 volume to 20 volumes fromthe viewpoint of heat generation control and the volume efficiency, morepreferably 4 volumes to 10 volumes from the viewpoint of the stirringefficiency.

The this step can be carried out by appropriately mixing the compoundrepresented by the General Formula (3), the base, and the condensingagent or the acid halogenating agent together. From the viewpoint of theyield, the compound represented by the General Formula (3) and the baseare preferably mixed together in advance.

The reaction temperature may be appropriately set. For example, thereaction temperature may be appropriately set within the range of −100°C. to 100° C. For allowing easy control of the temperature, ultralowtemperature conditions are preferably avoided. On the other hand, sincethe compound represented by the General Formula (4) is unstable at hightemperature, the reaction temperature is preferably −50° C. to 50° C.,more preferably −10° C. to 30° C.

The reaction time may be appropriately set within the range of, forexample, 5 minutes to 10 hours. Since, in general, the reaction becomescomplete in a short time after the mixing, the reaction time ispreferably 5 minutes to 5 hours, more preferably 15 minutes to 2 hours.

If necessary, post-treatment may be carried out after this reaction. Forexample, precipitated salt may be removed by filtration; water and awater-insoluble organic solvent may be added to perform solventextraction; and/or solvent extraction may be followed by removal of asolvent by concentration. In cases where the purity is insufficient,purification using a column may be carried out. However, since, evenwithout post-treatment or purification, the step (iii) described lateris hardly adversely affected, the reaction solution in the step (ii) ispreferably used as it is in the step (iii) from the viewpoint ofsimplifying the process.

<Step (iii)>

The step (iii) is described below.

The step (iii) is a step in which an azidation agent is allowed to acton a compound represented by General Formula (4):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted; R² represents aC₁-C₄ alkyl group; X represents —OP(O)(OPh)₂, —OP(O)(OEt)₂, —OC(O)OR³,or a halogen atom; and R³ represents a C₁-C₁₀ alkyl group which isoptionally substituted, a C₇-C₂₀ aralkyl group which is optionallysubstituted, or a C₆-C₁₂ aryl group which is optionally substituted),to obtain a compound represented by General Formula (5):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (4)).

The R¹ and the R² in the compound represented by the General Formula (4)have the same meanings as those in the description for the step (ii).

The R¹ and the R² in the compound represented by the General Formula (5)have the same meanings as those in the descriptions for the step (i) andthe step (ii).

Thus, specific examples of the compound represented by the GeneralFormula (5) include the following compounds.

Among the compounds mentioned above, the following compounds arepreferred as the compound represented by the General Formula (5).

The compound represented by the General Formula (5) may be either aracemic compound or a chiral compound. Since high optical purity isgenerally required in cases where the compound is produced as apharmaceutical or an intermediate therefor, the compound is preferably achiral compound having high optical purity. The optical purity ispreferably not less than 95.0% e.e., more preferably not less than 99.0%e.e., especially preferably not less than 99.5% e.e., most preferablynot less than 99.8% e.e.

Examples of the azidation agent include metal azides such as sodiumazide, potassium azide, and lithium azide; and trialkylsilyl azides suchas trimethylsilyl azide. Since trialkylsilyl azides may producetrialkylsilanol as a by-product, and an operation for its separationfrom the compound represented by General Formula (5) may be necessary,metal azides are preferred. Among the metal azides, sodium azide is morepreferred since it is easily available.

The amount of the azidation agent used may be appropriately set withinthe range of, for example, 0.7 molar equivalent to 10 molar equivalentswith respect to the amount of the compound represented by GeneralFormula (4). From the viewpoint of the yield and the cost, the amount ispreferably 1.0 molar equivalent to 5.0 molar equivalents, morepreferably 1.0 molar equivalent to 2.0 molar equivalents.

Examples of the reaction solvent include water; ketones such as acetone,2-butanone, methylisobutyl ketone, and cyclohexanone; aromatichydrocarbons such as benzene, toluene, xylene, chlorobenzene, andtrifluoromethylbenzene; aliphatic hydrocarbons such as n-pentane,n-hexane, n-heptane, and cyclohexane; esters such as methyl acetate,ethyl acetate, isopropyl acetate, and butyl acetate; ethers such asdiethyl ether, di-n-propyl ether, di-n-butyl ether, methyl isopropylether, methyl-tert-butyl ether, ethyl-tert-butyl ether, cyclopentylmethyl ether, tetrahydrofuran, dioxane, and 1,2-dimethoxyethane; amidessuch as N,N-dimethylformamide and N,N-dimethylacetamide; nitriles suchas acetonitrile and propionitrile; halogenated hydrocarbons such asdichloromethane, chloroform, carbon tetrachloride, and1,2-dichloroethane; and dimethylsulfoxide.

In cases where an alcohol is used as the reaction solvent, reaction withthe compound represented by General Formula (4) may occur to decreasethe yield. Among the reaction solvents described above, acetone,toluene, xylene, ethyl acetate, isopropyl acetate, and tetrahydrofuranare preferred from the viewpoints of the cost and availability, and ofsimplification of the process since the this step can be carried outcontinuously from the step (ii).

In particular, in cases where a metal azide is used as the azidationagent, and water is added to the solvent, the metal azide is dissolvedin the water, and the reaction efficiently proceeds, which is morepreferred. In particular, in cases where toluene or xylene is used incombination with water, the later-described post-treatment operationssuch as washing with water and extraction can be carried out withoutfurther addition of a solvent, which is still more preferred.

In cases where a metal azide is used as the azidation agent, and wateris used as the solvent, the compound represented by the General Formula(4) to be used as the material for the reaction is preferably a compoundwhich hardly undergoes degradation due to water. A compound representedby General Formula (4-1), in which the X is —OC(O)OR³, is morepreferably used.

The amount of the reaction solvent used may be appropriately set withinthe range of, for example, 0 volume to 100 volumes with respect to theamount of the compound represented by General Formula (4). The amount ofthe reaction solvent is preferably 1 volume to 20 volumes from theviewpoint of heat generation control and the volume efficiency, morepreferably 5 volumes to 10 volumes from the viewpoint of ease ofstirring.

The reaction temperature may be appropriately set within the range of,for example, −100° C. to 100° C. Since the temperature can be easilycontrolled at −30° C. to 50° C., ultralow temperature conditions arepreferably avoided. Further, since the compound represented by GeneralFormula (5) may cause a side reaction at a high temperature to decreasethe yield, the reaction temperature is more preferably −10° C. to 20° C.

The reaction time may be appropriately set within the range of, forexample, 5 minutes to 100 hours. Since the shelf stability of thecompound represented by General Formula (5) is not high even at a lowtemperature, the reaction time is preferably 1 hour to 40 hours, morepreferably 1 hour to 10 hours.

If necessary, post-treatment may be carried out after this reaction. Forexample, precipitated salt may be removed by filtration or washing withwater, and/or water and a water-insoluble organic solvent may be addedto perform extraction. In particular, in cases where an excessive amountof an azidation agent is used, it is sometimes risky to proceed to thenext step without removal of the azidation agent, which is explosive.Therefore, as an operation for sufficient removal of the azidation agentto the outside of the reaction system, washing with water is preferablycarried out using water and a water-insoluble solvent. In such a case,the step (iii) is more preferably carried out continuously, withoutfurther addition or substitution of a solvent, from the step (ii) whichis carried out in a water-insoluble solvent such as toluene or xylene,since, in this case, the washing with water can be carried out withoutfurther addition of a water-insoluble solvent, which is advantageousfrom the economical viewpoint. Since, in general, acid azide compoundssuch as the compounds represented by General Formula (5) are explosive,removal of the solvent by distillation should be avoided.

In Non-patent Document 1, in the step (ii), triethylamine is used as thebase, and diphenylphosphoryl azide is allowed to act as the condensingagent in 1,2-dichloroethane. That is, in Non-patent Document 1, acompound represented by General Formula (5) is obtained through acompound represented by General Formula (4) in which X is —OP(O)(OPh)₂,and the process of the step (ii) and the step (iii) is carried out inone step. The same applies to use of diethylphosphoryl azide instead ofdiphenylphosphoryl azide. However, in such a method, 1,2-dichloroethane,which is problematic in view of its toxicity and environmental load, isused as the solvent, and therefore the method is not suitable forindustrial-scale production. Thus, in industrial-scale production, it ispreferred to allow a chloroformic ester to act in the step (ii) using ahalogen-free solvent to obtain a compound represented by General Formula(4), and then to allow a metal azide to act in the step (iii), in astepwise manner as in the present invention. Since the combination of analkyl chloroformate and a metal azide is less expensive thandiphenylphosphoryl azide or diethylphosphoryl azide, the present methodis preferred also from the viewpoint of the cost.

<Step (iv)>

The step (iv) is described below.

The step (iv) is a method for producing a compound represented byGeneral Formula (6):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (5) below),which method comprises converting the azide group of a compoundrepresented by General Formula (5):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² represents aC₁-C₄ alkyl group) to an isocyanate group by Curtius rearrangementreaction, to obtain the compound represented by the General Formula (6).

R¹ and R² in the General Formulae (5) and (6) are the same as thosedescribed in the step (i).

Thus, specific examples of the compound represented by the GeneralFormula (6) include the following compounds.

Among the compounds mentioned above, the following compounds arepreferred as the compound represented by the General Formula (6).

The compound represented by the General Formula (6) may be either aracemic compound or a chiral compound. Since high optical purity isgenerally required in cases where the compound is produced as apharmaceutical or an intermediate therefor, the compound is preferably achiral compound having high optical purity. The optical purity ispreferably not less than 95.0% e.e., more preferably not less than 99.0%e.e., especially preferably not less than 99.5% e.e., most preferablynot less than 99.8% e.e.

The Curtius rearrangement is a reaction in which the compoundrepresented by General Formula (5), which is an acid azide compound, isheated to cause its conversion into an isocyanate compound, which isaccompanied by elimination of the nitrogen molecule in the azide group.The reaction can be allowed to proceed without addition of a reagent.

Examples of organic solvents which may be used in this step includeketones such as acetone, 2-butanone, methylisobutyl ketone, andcyclohexanone; aromatic hydrocarbons such as benzene, toluene, xylene,chlorobenzene, and trifluoromethylbenzene; aliphatic hydrocarbons suchas n-pentane, n-hexane, n-heptane, and cyclohexane; esters such asmethyl acetate, ethyl acetate, and isopropyl acetate; ethers such asdiethyl ether, di-n-propyl ether, di-n-butyl ether, methyl-tert-butylether, ethyl-tert-butyl ether, cyclopentyl methyl ether,tetrahydrofuran, dioxane, and 1,2-dimethoxyethane; amides such asN,N-dimethylformamide and N,N-dimethylacetamide; nitriles such asacetonitrile and propionitrile; halogenated hydrocarbons such asdichloromethane, chloroform, carbon tetrachloride, and1,2-dichloroethane; and dimethylsulfoxide.

Among these, benzene, toluene, xylene, chlorobenzene, andtrifluoromethylbenzene are preferred since they have high boilingpoints, and therefore the reaction rate can be increased by increasingthe reaction temperature, so that the processing time can be shortened.Toluene and xylene are more preferred since they can be usedcontinuously from the step (iii). That is, in cases where toluene orxylene is used, concentration and solvent replacement of the compoundrepresented by General Formula (5), which is explosive, can be avoided,and moreover, their use is economically advantageous. Water and alcoholsmay react with the compound represented by General Formula (6) toproduce, as a by-product, a dimer having a urea structure, or acarbamate, leading to a decrease in the yield.

The amount of the organic solvent used may be appropriately set withinthe range of, for example, 0 volume to 100 volumes with respect to theamount of the compound represented by General Formula (5). The amount ofthe organic solvent is preferably 3 volumes to 30 volumes from theviewpoint of heat generation control and the volume efficiency, morepreferably 7 volumes to 15 volumes.

The reaction temperature may be appropriately set within the range of,for example, 0° C. to 300° C. From the viewpoint of reducing thereaction time and preventing degradation of the compound represented byGeneral Formula (6), the reaction temperature is preferably 40° C. to150° C., more preferably 70° C. to 110° C. In cases where the reactiontemperature is higher than the temperature described above, the compoundrepresented by General Formula (6) produced tends to be degraded,resulting in a low yield. On the other hand, in cases where the reactiontemperature is lower than the temperature described above, the compoundrepresented by General Formula (5) may accumulate in the reaction systemwithout undergoing Curtius rearrangement, and the subsequent temperatureincrease may cause the reaction to proceed rapidly, resulting ingeneration of a large amount of nitrogen in a short time. Thus, from theviewpoint of safety, it is preferred, if necessary, to control theamount of nitrogen generated.

Since nitrogen is generated as the reaction proceeds in this step, theamount of nitrogen generated needs to be controlled from the viewpointof safety, especially in industrial-scale production. Therefore, it ispreferred to add the compound represented by General Formula (5)dropwise to a solvent warmed to a temperature at which Curtiusrearrangement of the compound represented by General Formula (5)immediately occurs, and to control the amount of nitrogen generatedbased on the amount of the compound added dropwise.

From the viewpoint of the safety described in the previous section, thetemperature is ideally set such that the reaction is completedimmediately after the addition of the compound represented by GeneralFormula (5). Practically, however, in order to complete the reaction,heating is preferably continued for 0 hour to 5 hours after the additionof the compound represented by General Formula (5). The heating is morepreferably continued for 0 hour to 3 hours.

If necessary, post-treatment may be carried out after this reaction. Forexample, salt may be removed by filtration; water may be added toperform solvent extraction; and/or a solvent may be removed byconcentration. Depending on the purity, purification using a column maybe carried out. However, since the compound represented by GeneralFormula (6) easily reacts with water to produce a dimer having a ureastructure, post-treatment is preferably avoided.

In Non-patent Document 1, instead of carrying out the step (ii),reaction is performed under heat in 1,2-dichloroethane usingtriethylamine as the base and diphenylphosphoryl azide as the condensingagent. In such a method, a compound represented by General Formula (5)is obtained, but, practically, since the reaction is carried out underheat, the compound represented by General Formula (5) immediatelyundergoes Curtius rearrangement, and is converted into a compoundrepresented by General Formula (6). That is, by the method described inNon-patent Document 1, the steps (ii), (iii), and (iv) can be obtainedin one step, and the same applies to use of diethylphosphoryl azideinstead of diphenylphosphoryl azide. However, in this method, diphenylphosphate and diethyl phosphate are produced as by-products, so that anoperation for removing these by-products is necessary. In Non-patentDocument 1, purification is carried out by flash column chromatography.However, this operation is not suitable for industrial-scale production.As an alternative to this purification, washing with water may bepossible. However, when the compound represented by General Formula (6)is brought into contact with water, a dimer having a urea structure isproduced as a by-product as described above, and diphenyl phosphate anddiethyl phosphate produced as by-products tend to cause side reactionswith the compound represented by General Formula (6), resulting in a lowyield.

Therefore, from the viewpoint of both quality and yield, the steps (ii)to (iv) are preferably carried out in a stepwise manner as describedabove such that, for example, a chloroformic ester is allowed to act toobtain a compound represented by General Formula (4) in the step (ii); ametal azide is allowed to act to obtain a compound represented byGeneral Formula (5) in the step (iii); and the compound represented byGeneral Formula (5) is converted to a compound represented by GeneralFormula (6) by Curtius rearrangement in the step (iv).

After the steps (i) to (iv), the compound represented by General Formula(6) can be converted to an α-substituted cysteine represented by GeneralFormula (1) or a salt thereof. The method for this conversion is notlimited, and the conversion is preferably carried out by a methodcomprising the step (v) described below.

<Step (v)>

The step (v) is described below.

The step (v) is a step for obtaining an α-substituted cysteinerepresented by General Formula (1):

(wherein R² represents a C₁-C₄ alkyl group)or a salt thereof, which step comprises the processes of:

(a) converting the isocyanate group in a compound represented by GeneralFormula (6):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² has the samemeaning as in the General Formula (1)) to an amino group;

(b) hydrolyzing the ester group; and

(c) removing the tert-butyl group by action of an acid.

In the compounds represented by the General Formulae (1) and (6), the R¹and the R² are the same as those described in the step (i).

Thus, specific examples of the α-substituted cysteine represented by theGeneral Formula (1) include the following compounds.

Among the compounds mentioned above, the following compounds arepreferred as the α-substituted cysteine represented by the GeneralFormula (1).

The α-substituted cysteine represented by General Formula (1) may be inthe form of a salt. Examples of the salt of the α-substituted cysteineinclude inorganic acid salts such as hydrochloric acid salt, sulfuricacid salt, nitric acid salt, and phosphoric acid salt; carboxylic acidsalts such as acetic acid salt, propionic acid salt, oxalic acid salt,malic acid salt, maleic acid salt, citric acid salt, succinic acid salt,tartaric acid salt, and mandelic acid salt; sulfonic acid salts such asmethanesulfonic acid salt, p-toluenesulfonic acid salt, andcamphorsulfonic acid salt; alkali metal salts such as sodium salt andpotassium salt; alkaline earth metal salts such as calcium salt,magnesium salt, and barium salt; and organic amine salts such asammonium salt, trimethylamine salt, triethylamine salt, phenethylaminesalt, and pyridine salt. Among these, hydrochloric acid salt ispreferred.

The compound represented by the General Formula (1) may be either aracemic compound or a chiral compound. Since high optical purity isgenerally required in cases where the compound is produced as apharmaceutical or an intermediate therefor, the compound is preferably achiral compound having high optical purity. The optical purity ispreferably not less than 95.0% e.e., more preferably not less than 99.0%e.e., especially preferably not less than 99.5% e.e., most preferablynot less than 99.8% e.e.

(a) As the reaction to be carried out in the process of converting theisocyanate group to an amino group, a known method may be used, ifappropriate. Specific examples of the method include a method in whichan acid or a base is allowed to act to cause direct conversion to theamino group, and a method in which an alcohol is allowed to act to causeconversion to a carbamate, followed by its conversion to the amino groupusing an acid or a base, or by catalytic reduction.

Examples of the acid or the base to be used in the method by directconversion to the amino group include inorganic acids such ashydrochloric acid and sulfuric acid; and alkali metal hydroxides such assodium hydroxide and potassium hydroxide. In these cases, the reactiontemperature is usually 0° C. to 200° C., and the reaction time isusually 30 minutes to 50 hours.

Examples of the method carried out through a carbamate include a methodin which benzyl alcohol, p-methoxybenzyl alcohol, or the like is allowedto act to cause conversion to a benzyloxycarbonyl-protected amino group,followed by deprotection of the benzyloxycarbonyl group by catalyticreduction, and a method in which tert-butyl alcohol is allowed to act tocause conversion to a tert-butoxycarbonyl (hereinafter referred to as“Boc”)-protected amino group, followed by allowing an acid to act fordeprotection of the Boc group.

(b) In the process of hydrolyzing the ester group, water, and an acid ora base is allowed to act on the compound represented by the GeneralFormula (6).

In cases where the hydrolysis is carried out by action of a base,examples of the base include alkali metal hydroxides such as sodiumhydroxide, lithium hydroxide, and potassium hydroxide; and alkali metalcarbonates such as lithium carbonate, sodium carbonate, and potassiumcarbonate.

In cases where the hydrolysis is carried out by action of an acid,examples of the acid include inorganic acids such as hydrochloric acid,sulfuric acid, phosphoric acid, hydrogen bromide, hydrogen fluoride, andhydrogen iodide; and organic acids such as trifluoroacetic acid,trifluoromethanesulfonic acid, methanesulfonic acid, andp-toluenesulfonic acid.

Both in the cases where the base is allowed to act, and in the caseswhere the acid is allowed to act, the reaction temperature is usually 0°C. to 200° C., and the reaction time is usually 30 minutes to 50 hours.

(c) In the process of removing the tert-butyl group by action of anacid, an acid is allowed to act on the compound represented by GeneralFormula (6).

The acid to be used is not limited as long as the removal of thetert-butyl group can be achieved therewith, and is preferably a strongacid. Examples of the acid include inorganic acids such as hydrochloricacid, sulfuric acid, phosphoric acid, hydrogen bromide, hydrogenfluoride, and hydrogen iodide; and organic acids such as trifluoroaceticacid, trifluoromethanesulfonic acid, methanesulfonic acid, andp-toluenesulfonic acid. Among these, hydrochloric acid and sulfuric acidare preferred since these are inexpensive.

In such cases, the reaction temperature is usually 50° C. to 200° C.,and the reaction time is usually 10 hours to 150 hours.

The above-described three processes: (a) converting the isocyanate groupto an amino group, (b) hydrolyzing the ester group, and (c) removing thetert-butyl group, may be carried out in any order, and two or moreprocesses may be carried out at the same time.

The step (v) is preferably carried out through the steps (vi-1), (vi-2),and (vii) described below. By allowing the reaction to proceed throughthe step (vi-1), elimination of the tert-butyl group, which generallyrequires a long time, can be allowed to proceed quickly to obtain acompound represented by General Formula (7-1):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (6)).

Further, by allowing the reaction to proceed through the step (vi-2), acompound represented by General Formula (7-2):

(wherein R² has the same meaning as R² in the General Formula (6)) canbe obtained. Since the compound represented by the General Formula (7-2)has high crystallinity, and low solubility in water at a pH of not morethan the point of neutralization and in nonpolar solvents, bothwater-soluble impurities including inorganic salts and water-insolubleimpurities including reaction intermediates can be easily removed bycrystallization. Thus, the α-substituted cysteine represented by GeneralFormula (1) or the salt thereof obtained after the subsequent step (vii)can be obtained with high purity.<Steps (vi-1) and (vi-2)>

The steps (vi-1) and (vi-2) are described below.

The step (vi-1) is a step of allowing an acid to act on a compoundrepresented by General Formula (6):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² represents aC₁-C₄ alkyl group)to construct a thiazolidinone ring, thereby obtaining a compoundrepresented by General Formula (7-1):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (6)).

The R¹ and the R² in the compounds represented by the General Formulae(6) and (7-1) are the same as those described in the step (i).

Thus, specific examples of the compound represented by General Formula(7-1) include the following compounds.

Among the compounds mentioned above, the following compounds arepreferred as the compound represented by the General Formula (7-1).

The compound represented by the General Formula (7-1) may be either aracemic compound or a chiral compound. Since high optical purity isgenerally required in cases where the compound is produced as apharmaceutical or an intermediate therefor, the compound is preferably achiral compound having high optical purity. The optical purity ispreferably not less than 95.0% e.e., more preferably not less than 99.0%e.e., especially preferably not less than 99.5% e.e., most preferablynot less than 99.8% e.e.

Examples of the acid used in the step (vi-1) include inorganic acidssuch as hydrochloric acid, sulfuric acid, phosphoric acid, hydrogenbromide, hydrogen fluoride, and hydrogen iodide; and organic acids suchas trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonicacid, and p-toluenesulfonic acid. Among these, hydrochloric acid,sulfuric acid, phosphoric acid, and p-toluenesulfonic acid are morepreferred from the viewpoint of the cost.

The amount of the acid used may be appropriately set within the rangeof, for example, 0.01 molar equivalent to 50 molar equivalents withrespect to the amount of the compound represented by General Formula(6). Considering the cost, and laboriousness of the post-treatment, theamount is preferably 0.1 molar equivalent to 10 molar equivalents, morepreferably 0.5 molar equivalent to 3 molar equivalents.

Examples of the reaction solvent to be used include ketones such asacetone, 2-butanone, and methylisobutyl ketone; aromatic hydrocarbonssuch as benzene, toluene, xylene, chlorobenzene, andtrifluoromethylbenzene; aliphatic hydrocarbons such as n-pentane,n-hexane, n-heptane, and cyclohexane; esters such as methyl acetate,ethyl acetate, and isopropyl acetate; ethers such as diethyl ether,di-n-propyl ether, di-n-butyl ether, methyl isopropyl ether,methyl-tert-butyl ether, ethyl-tert-butyl ether, cyclopentyl methylether, tetrahydrofuran, dioxane, and 1,2-dimethoxyethane; amides such asN,N-dimethylformamide and N,N-dimethylacetamide; nitriles such asacetonitrile and propionitrile; halogenated hydrocarbons such asdichloromethane, chloroform, carbon tetrachloride, and1,2-dichloroethane; and dimethylsulfoxide. In cases where water or analcohol is used, reaction with the compound represented by GeneralFormula (6) tends to occur to produce a dimer having a urea structure ora carbamate as a by-product, leading to a decrease in the yield. Thus,use of water or an alcohol as a main solvent for the step (vi-1) is notpreferred. In cases where water or an alcohol is used for dissolving aninorganic acid or an inorganic base, the amount of the water or thealcohol used is preferably minimum.

Thus, among the reaction solvents described above, aromatic hydrocarbonssuch as benzene, toluene, xylene, chlorobenzene, andtrifluoromethylbenzene; aliphatic hydrocarbons such as n-pentane,n-hexane, n-heptane, and cyclohexane; and halogenated hydrocarbons suchas dichloromethane, chloroform, carbon tetrachloride, and1,2-dichloroethane; are preferred from the viewpoint of stabilityagainst acids. It is more preferred to carry out the this step usingtoluene or xylene continuously from the step (iv) without changing thesolvent, from the viewpoint of the cost.

The reaction temperature may be appropriately set within the range of,for example, −30° C. to 100° C. However, since, in cases where thereaction is carried out within the range of 70° C. to 100° C., thecompound represented by General Formula (7-1) produced is converted tothe compound represented by General Formula (7-2) in a short time. Thus,in cases where the compound represented by General Formula (7-1) is tobe isolated, the reaction is carried out preferably within the range of−30° C. to 50° C., more preferably within the range of 0° C. to 30° C.

The reaction time may be appropriately set within the range of, forexample, 0.5 hour to 20 hours. In cases where the reaction is carriedout for a reaction time within the range of 10 hours to 20 hours, thecompound represented by General Formula (7-1) produced tends to beconverted to the compound represented by General Formula (7-2) dependingon the temperature setting. Thus, in cases where the compoundrepresented by General Formula (7-1) is to be isolated, the reactiontime is preferably 0.5 hour to 10 hours, more preferably 0.5 hour to 3hours.

If necessary, post-treatment may be carried out. For example,neutralization may be carried out by adding a base; water and awater-insoluble organic solvent may be added to perform extraction;and/or, in cases where the acid is volatile, it may be removed byconcentration. From the viewpoint of simplification of the process, itis preferred to proceed to the step (vi-2) described below withoutcarrying out post-treatment.

The step (vi-2) is a step of allowing an acid or a base to act on acompound represented by General Formula (7-1):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (6)) to hydrolyze the ester group, and thereby obtain a compoundrepresented by General Formula (7-2):

(wherein R² has the same meaning as R² in the General Formula (6)).

The R² in the General Formulae (7-1) and (7-2) are the same as thatdescribed in the step (i).

Thus, specific examples of the compound represented by the GeneralFormula (7-2) include the following compounds.

Among the compounds mentioned above, the following compounds arepreferred as the compound represented by the General Formula (7-2).

The compound represented by the General Formula (7-2) may be either aracemic compound or a chiral compound. Since high optical purity isgenerally required in cases where the compound is produced as apharmaceutical or an intermediate therefor, the compound is preferably achiral compound having high optical purity. The optical purity ispreferably not less than 95.0% e.e., more preferably not less than 99.0%e.e., especially preferably not less than 99.5% e.e., most preferablynot less than 99.8% e.e.

Examples of the acid used in the step (vi-2) include inorganic acidssuch as hydrochloric acid, sulfuric acid, phosphoric acid, hydrogenbromide, hydrogen fluoride, and hydrogen iodide; and organic acids suchas trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonicacid, and p-toluenesulfonic acid. Among these, hydrochloric acid,sulfuric acid, and phosphoric acid are more preferred from the viewpointof the cost. Hydrochloric acid is still more preferred since it is avolatile acid and can be easily removed by concentration.

Alternatively, a base may be allowed to act to perform the hydrolysis.Examples of the base to be used include alkali metal hydroxides such aslithium hydroxide, sodium hydroxide, and potassium hydroxide; and alkalimetal carbonates such as sodium carbonate, lithium carbonate, andpotassium carbonate.

In cases where a base is used, its recovery by filtration needs to becarried out after adjustment of the pH by addition of an acid since, asdescribed above, the compound represented by the General Formula (7-2)has low solubility in acidic water, but has high solubility in basicwater. Since this is accompanied by the risk of contamination with aninorganic salt generated during the neutralization, an acid ispreferably used from the viewpoint of quality.

Since an acid is also used in the step (vi-1), the acid used in the step(vi-1) may be allowed to act continuously without replacement, tocontinuously carry out the step (vi-1) and the step (vi-2).Alternatively, an acid may be further added in the step (vi-2). In sucha case, the acid may be either the same as, or different from, the acidused in the step (vi-1). However, from the viewpoint of simplificationof the process, it is preferred to allow the acid used in the step(vi-1) to act continuously without carrying out post-treatment after thestep (vi-1), to carry out these steps continuously. That is, thereaction solution containing the compound represented by the GeneralFormula (7-1) obtained in the step (vi-1) is preferably used in the step(vi-2) as it is after, for example, changing the reaction temperature.

In cases where an acid is used, the amount of the acid used may beappropriately set within the range of 0.01 molar equivalent to 50 molarequivalents with respect to the amount of the compound represented byGeneral Formula (7-1). Considering the cost, and laboriousness of thepost-treatment, the amount is preferably 0.1 molar equivalent to 10molar equivalents, more preferably 0.5 molar equivalent to 2 molarequivalents.

In cases where an acid is used, examples of the reaction solvent includewater; ketones such as acetone, 2-butanone, and methylisobutyl ketone;aromatic hydrocarbons such as benzene, toluene, xylene, chlorobenzene,and trifluoromethylbenzene; aliphatic hydrocarbons such as n-pentane,n-hexane, n-heptane, and cyclohexane; esters such as methyl acetate,ethyl acetate, and isopropyl acetate; ethers such as diethyl ether,di-n-propyl ether, di-n-butyl ether, methyl isopropyl ether,methyl-tert-butyl ether, ethyl-tert-butyl ether, cyclopentyl methylether, tetrahydrofuran, dioxane, and 1,2-dimethoxyethane; amides such asN,N-dimethylformamide and N,N-dimethylacetamide; nitriles such asacetonitrile and propionitrile; halogenated hydrocarbons such asdichloromethane, chloroform, carbon tetrachloride, and1,2-dichloroethane; and dimethylsulfoxide.

Since alcohols tend to inhibit the progress of the hydrolysis, aromatichydrocarbons such as benzene, toluene, xylene, chlorobenzene, andtrifluoromethylbenzene; aliphatic hydrocarbons such as n-pentane,n-hexane, n-heptane, and cyclohexane; and halogenated hydrocarbons suchas dichloromethane, chloroform, carbon tetrachloride, and1,2-dichloroethane; are preferred among the reaction solvents describedabove, from the viewpoint of stability against acids. Benzene, toluene,xylene, chlorobenzene, and trifluoromethylbenzene are more preferredsince these allow heating at high temperature. Most preferably, tolueneor xylene is used continuously from the step (vi-1), from the viewpointof the cost.

In cases where an acid is used, the amount of the solvent used may beappropriately set within the range of, for example, 0 volume to 100volumes with respect to the amount of the compound represented byGeneral Formula (6). From the viewpoint of the volume efficiency, theamount is preferably 0 volume to 20 volumes, more preferably 0 volume to5 volumes.

In cases where an acid is used, the reaction temperature may beappropriately set within the range of, for example, −30° C. to 200° C.Considering improvement of the degree of conversion and the boilingpoint of the solvent, the reaction temperature is preferably 20° C. to120° C. In cases where the temperature is too high, degradation of thecompound represented by General Formula (7-2) proceeds, leading to adecrease in the yield. Thus, the reaction temperature is more preferably50° C. to 90° C.

In cases where an acid is used, the reaction time may be appropriatelyset within the range of, for example, 0 hour to 100 hours. Depending onthe temperature, in cases where the reaction is carried out for a longtime, degradation of the compound represented by General Formula (7-2)proceeds, leading to a decrease in the yield. Therefore, the reactiontime is preferably 0 hour to 30 hours. From the viewpoint of improvementof the degree of conversion, the reaction time is more preferably 5hours to 30 hours.

In cases where an acid is used, examples of the post-treatment after thereaction include a method in which the compound represented by GeneralFormula (7-2) is recovered from the reaction solvent by crystallizationtaking advantage of the fact that the compound is solid in an acidicsolution. Alternatively, if necessary, the solvent may be simply removedby concentration; the reaction solution may be neutralized using an acidor a base, and salt may be separated by adsorption of the product ofinterest to an ion-exchange resin, followed by recovering the product bycrystallization; the acid or the base may be removed by adsorption to anion-exchange resin, followed by recovering the compound from the eluateby crystallization; or, depending on the purity, purification may becarried out using a column or by recrystallization. In cases where onlyan acid is used, the compound represented by General Formula (7-2)generally has high crystallinity, and can be recovered with high purity.Therefore, it is preferred to recover the compound from the reactionsolvent by performing only crystallization.

In cases where a base is used, examples of the reaction solvent include,from the viewpoint of stability against the base, water; ketones such asacetone, 2-butanone, and methylisobutyl ketone; alcohols such asmethanol, ethanol, n-propyl alcohol, isopropyl alcohol, n-butanol, andtert-butanol; aromatic hydrocarbons such as benzene, toluene, xylene,chlorobenzene, and trifluoromethylbenzene; aliphatic hydrocarbons suchas n-pentane, n-hexane, n-heptane, and cyclohexane; esters such asmethyl acetate, ethyl acetate, and isopropyl acetate; ethers such asdiethyl ether, di-n-propyl ether, di-n-butyl ether, methyl isopropylether, methyl-tert-butyl ether, ethyl-tert-butyl ether, cyclopentylmethyl ether, tetrahydrofuran, dioxane, and 1,2-dimethoxyethane; amidessuch as N,N-dimethylformamide and N,N-dimethylacetamide; nitriles suchas acetonitrile and propionitrile; halogenated hydrocarbons such asdichloromethane, chloroform, carbon tetrachloride, and1,2-dichloroethane; and dimethylsulfoxide. In particular, preferredexamples of the reaction solvent include aromatic hydrocarbons such asbenzene, toluene, xylene, chlorobenzene, and trifluoromethylbenzene;aliphatic hydrocarbons such as n-pentane, n-hexane, n-heptane, andcyclohexane; and ethers such as diethyl ether, di-n-propyl ether,di-n-butyl ether, methyl isopropyl ether, methyl-tert-butyl ether,ethyl-tert-butyl ether, cyclopentyl methyl ether, tetrahydrofuran,dioxane, and 1,2-dimethoxyethane. Among these, benzene, toluene, xylene,chlorobenzene, and trifluoromethylbenzene are more preferred since theseallow heating at high temperature. Most preferably, toluene or xylene isused continuously from the step (vi-1) from the viewpoint of the cost.

In cases where a base is used, the amount of the base may beappropriately set within the range of 0.9 molar equivalent to 20 molarequivalents with respect to the amount of the compound represented byGeneral Formula (7-1). Considering the cost, and laboriousness of thepost-treatment, the amount is preferably 0.9 molar equivalent to 5.0molar equivalents, more preferably 1.0 molar equivalent to 3.0 molarequivalents. In cases where no post-treatment is carried out after thestep (vi-1), and the acid used in the step (vi-1) is continuouslyallowed to act to carry out the this step continuously from the step(vi-1), a base required for neutralization of the acid may be furtheradded.

In cases where a base is used, the amount of the solvent used may beappropriately set within the range of, for example, 0 volume to 100volumes with respect to the amount of the compound represented byGeneral Formula (6). From the viewpoint of the volume efficiency, theamount is preferably 0 volume to 20 volumes, more preferably 0 volume to5 volumes.

In cases where a base is used, the reaction temperature may beappropriately set within the range of, for example, −30° C. to 100° C.For suppression of side reactions, the reaction temperature ispreferably 0° C. to 80° C. In cases where the temperature is too high,degradation of the compound represented by General Formula (7-2) mayproceed, leading to a decrease in the yield. Thus, the reactiontemperature is more preferably 0° C. to 50° C.

In cases where a base is used, the reaction time may be appropriatelyset within the range of, for example, 0 hour to 100 hours. Depending onthe temperature, in cases where the reaction is carried out for a longtime, degradation of the compound represented by General Formula (7-2)may proceed, leading to a decrease in the yield. Therefore, the reactiontime is preferably 0 hour to 30 hours. From the viewpoint of improvementof the degree of conversion, the reaction time is more preferably 5hours to 30 hours.

In cases where a base is used, this reaction may be followed by apost-treatment in which the compound represented by General Formula(7-2) is recovered from the reaction solvent by crystallization takingadvantage of the fact that the compound is solid in an acidic solution.More specifically, the base may be neutralized with an acid, and then anacid may be further added. Alternatively, the solvent may be simplyremoved by concentration; the reaction solution may be neutralized usingan acid, and salt may be separated by adsorption of the product ofinterest to an ion-exchange resin, followed by recovering the product bycrystallization; or the base may be removed by adsorption to anion-exchange resin, followed by recovering the product of interest fromthe eluate by crystallization. Depending on the purity, purification maybe carried out using a column or by recrystallization. However, in caseswhere only an acid is used, the compound represented by General Formula(7-2) generally has high crystallinity, and can be recovered with highpurity. Therefore, it is preferred to recover the compound from thereaction solvent by performing only crystallization.

Among the compounds represented by General Formula (7-2), GeneralFormula (7S-2):

(wherein R² has the same meaning as R² in the General Formula (7-2))

and General Formula (7R-2):

(wherein R² has the same meaning as R² in the General Formula (7-2))have higher crystallinity than racemic compounds. Accordingly,improvement of the optical purity by crystallization or the like can bemore easily carried out with a chiral compound wherein either one ofGeneral Formula (7S-2) or General Formula (7R-2) is selectivelycontained, compared to a racemic compound.

In cases where the optical purity of the compound represented by GeneralFormula (7-2) does not reach a desired level after carrying out thepost-treatment described above, recrystallization may be repeated toincrease the optical purity. In particular, in cases where the compoundrepresented by General Formula (1) in the step (vii) described later isa chiral compound, the optical purity of the compound represented byGeneral Formula (7-2) as a material is preferably preliminarilyincreased to a desired level by crystallization.

Examples of the crystallization solvent for the compound represented byGeneral Formula (7-2) include water; alcohols such as methanol, ethanol,n-propyl alcohol, isopropyl alcohol, n-butanol, and tert-butanol;ketones such as acetone, 2-butanone, and methylisobutyl ketone; aromatichydrocarbons such as benzene, toluene, xylene, chlorobenzene, andtrifluoromethylbenzene; aliphatic hydrocarbons such as n-pentane,n-hexane, n-heptane, and cyclohexane; esters such as methyl acetate,ethyl acetate, and isopropyl acetate; ethers such as diethyl ether,di-n-propyl ether, di-n-butyl ether, methyl isopropyl ether,methyl-tert-butyl ether, ethyl-tert-butyl ether, cyclopentyl methylether, tetrahydrofuran, dioxane, and 1,2-dimethoxyethane; amides such asN,N-dimethylformamide and N,N-dimethylacetamide; nitriles such asacetonitrile and propionitrile; halogenated hydrocarbons such asdichloromethane, chloroform, carbon tetrachloride, and1,2-dichloroethane; and dimethylsulfoxide. One of these solvents may beused, or two or more of the solvents may be used as a mixture.

In particular, preferred examples of the crystallization solvent includepolar solvents, for example, alcohols such as methanol, ethanol,n-propyl alcohol, isopropyl alcohol, n-butanol, and tert-butanol;ketones such as acetone, 2-butanone, and methylisobutyl ketone; esterssuch as methyl acetate, ethyl acetate, and isopropyl acetate; amidessuch as N,N-dimethylformamide and N,N-dimethylacetamide; nitriles suchas acetonitrile and propionitrile; and dimethylsulfoxide; since theseshow high solubility and high purification efficiency. Among these,alcohols such as methanol, ethanol, n-propyl alcohol, isopropyl alcohol,n-butanol, and tert-butanol; ketones such as acetone, 2-butanone, andmethylisobutyl ketone; esters such as methyl acetate, ethyl acetate, andisopropyl acetate; and nitriles such as acetonitrile and propionitrile;are more preferred since these have low boiling points, and can beeasily removed by vacuum drying.

For improvement of the recovery, it is preferred to add, as a poorsolvent(s), one or more of low-polarity solvents to these polarsolvents. Examples of the low-polarity solvents include aromatichydrocarbons such as benzene, toluene, xylene, chlorobenzene, andtrifluoromethylbenzene; and aliphatic hydrocarbons such as n-pentane,n-hexane, n-heptane, and cyclohexane.

<Step (vii)>

The step (vii) is described below.

The step (vii) is a step of allowing an acid or a base to act on acompound represented by General Formula (7-2):

(wherein R² represents a C₁-C₄ alkyl group)to open the thiazolidinone ring, and thereby obtain an α-substitutedcysteine represented by General Formula (1):

(wherein R² represents a C₁-C₄ alkyl group)or the salt thereof.

Examples of the acid used in the step (vii) include inorganic acids suchas hydrochloric acid, sulfuric acid, phosphoric acid, hydrogen bromide,hydrogen fluoride, and hydrogen iodide; and organic acids such astrifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonicacid, and p-toluenesulfonic acid.

Examples of the base include alkali metal hydroxides such as lithiumhydroxide, sodium hydroxide, and potassium hydroxide; and alkali metalcarbonates such as sodium carbonate, lithium carbonate, and potassiumcarbonate. Among these, strong bases are preferred. Specific examples ofthe strong bases include alkali metal hydroxides such as lithiumhydroxide, sodium hydroxide, and potassium hydroxide.

In cases where a base is used, dimerization of the resultingα-substituted cysteine easily occurs to form a disulfide. Therefore, anacid is preferably used from the viewpoint of the yield and the quality.In particular, volatile acids such as hydrochloric acid, trifluoroaceticacid, hydrogen bromide, hydrogen fluoride, and hydrogen iodide are morepreferred since these can be removed by performing only concentration.From the viewpoint of the cost, hydrochloric acid is most preferred.

The acid or the base used in the step (vi-2) can also be used as atleast part of the acid or the base used in the step (vii). That is, bysubjecting the reaction solution obtained in the step (vi-2) as it is tothe reaction conditions for the later-described step (vii) withoutcarrying out post-treatment after the step (vi-2), an α-substitutedcysteine represented by General Formula (1):

(wherein R² represents a C₁-C₄ alkyl group)or a salt thereof can be obtained.

However, in cases where the α-substituted cysteine represented byGeneral Formula (1) or the salt thereof is to be produced with highpurity, the compound represented by General Formula (7-2), which is usedas a material, also needs to be highly pure. Thus, from the viewpoint ofquality control, it is preferred to carry out, as described above, theoperation of recovering the compound represented by General Formula(7-2) after the step (vi-2) by crystallization to remove impurities,followed by carrying out the step (vii).

The amount of the acid or the base used may be appropriately set withinthe range of, for example, 0.1 molar equivalent to 200 molar equivalentswith respect to the amount of the compound represented by GeneralFormula (7). From the viewpoint of the volume efficiency, the amount ispreferably 1 molar equivalent to 30 molar equivalents. In cases where anacid is used, the compound represented by General Formula (7) ispreferably completely dissolved in the acid from the viewpoint ofreducing the reaction time. Considering the load of the process ofremoving the acid, the amount of the acid is more preferably 5 molarequivalents to 20 molar equivalents.

The reaction may be carried out in the presence of a solvent, ifnecessary. Examples of the solvent include water; alcohols such asmethanol, ethanol, n-propyl alcohol, isopropyl alcohol, and n-butanol;ketones such as acetone, 2-butanone, and methylisobutyl ketone; aromatichydrocarbons such as benzene, toluene, xylene, chlorobenzene, andtrifluoromethylbenzene; aliphatic hydrocarbons such as n-pentane,n-hexane, n-heptane, and cyclohexane; esters such as methyl acetate,ethyl acetate, and isopropyl acetate; ethers such as diethyl ether,di-n-propyl ether, di-n-butyl ether, methyl isopropyl ether,methyl-tert-butyl ether, ethyl-tert-butyl ether, cyclopentyl methylether, tetrahydrofuran, dioxane, and 1,2-dimethoxyethane; amides such asN,N-dimethylformamide and N,N-dimethylacetamide; nitriles such asacetonitrile and propionitrile; halogenated hydrocarbons such asdichloromethane, chloroform, carbon tetrachloride, and1,2-dichloroethane; and dimethylsulfoxide. In cases where an acid isused, the solvent is preferably one which does not cause a side reactionwith the acid. Aromatic hydrocarbons such as benzene, toluene, xylene,chlorobenzene, and trifluoromethylbenzene; and aliphatic hydrocarbonssuch as n-pentane, n-hexane, n-heptane, and cyclohexane; are preferred.However, since, as the concentration of the acid increases, the reactionis better promoted, it is more preferred to use the acid as a solventwithout using another solvent.

The reaction temperature may be appropriately set within the range of,for example, −30° C. to 300° C. Considering the boiling point of thesolvent, the reaction temperature is preferably 25° C. to 150° C. Inparticular, in cases where a volatile acid is used, the reactiontemperature is more preferably 80° C. to 110° C., since the acidvolatilizes and the acid concentration decreases when the reactiontemperature exceeds the boiling point, resulting in a decrease in thereaction rate. It is also possible to carry out the reaction using asealable, pressure-resistant reaction vessel while preventingvolatilization of the acid. In such cases, the preferred range of thereaction temperature is not limited to the range described above.

The reaction time may be appropriately set within the range of, forexample, 1 hour to 300 hours. From the viewpoint of the yield, thereaction time is preferably 10 hours to 200 hours. In cases where anacid is used, the reaction time is more preferably 40 hours to 100hours.

In cases where an acid is used, the post-treatment of the reaction maybe appropriately selected depending on whether the α-substitutedcysteine is recovered as a salt or not. For example, in cases where theα-substituted cysteine is recovered as a salt with an acid, the solventmay be removed by concentration. In cases where the α-substitutedcysteine is recovered as an α-substituted cysteine, a base may be addedto the reaction solution to the point of neutralization to precipitatethe α-substituted cysteine, followed by recovering the α-substitutedcysteine by filtration. Alternatively, the reaction solution may bepassed through a cation-exchange column to adsorb the α-substitutedcysteine, thereby removing the acid, and the eluate containing theα-substituted cysteine may then be concentrated to recover theα-substituted cysteine. For obtaining an α-substituted cysteine or asalt thereof with high purity, recrystallization is preferably carriedout to increase the purity.

In cases where the recrystallization is carried out, examples of thecrystallization solvent include water; alcohols such as methanol,ethanol, n-propyl alcohol, isopropyl alcohol, n-butanol, andtert-butanol; ketones such as acetone, 2-butanone, and methylisobutylketone; aromatic hydrocarbons such as benzene, toluene, xylene,chlorobenzene, and trifluoromethylbenzene; aliphatic hydrocarbons suchas n-pentane, n-hexane, n-heptane, and cyclohexane; esters such asmethyl acetate, ethyl acetate, and isopropyl acetate; ethers such asdiethyl ether, di-n-propyl ether, di-n-butyl ether, methyl isopropylether, methyl-tert-butyl ether, ethyl-tert-butyl ether, cyclopentylmethyl ether, tetrahydrofuran, dioxane, and 1,2-dimethoxyethane; amidessuch as N,N-dimethylformamide and N,N-dimethylacetamide; nitriles suchas acetonitrile and propionitrile; halogenated hydrocarbons such asdichloromethane, chloroform, carbon tetrachloride, and1,2-dichloroethane; and dimethylsulfoxide. One of these solvents may beused, or two or more of the solvents may be used as a mixture. Alcoholssuch as methanol, ethanol, n-propyl alcohol, isopropyl alcohol,n-butanol, and tert-butanol; and ketones such as acetone, 2-butanone,and methylisobutyl ketone; are preferred since the purification can becarried out effectively with these.

Since an α-substituted cysteine is highly soluble in these solvents, andcrystallization in a single solvent results in a low recovery, it ismore preferred to add, as a poor solvent(s), one or more of nonpolarsolvents such as aromatic hydrocarbons including benzene, toluene,xylene, chlorobenzene, and trifluoromethylbenzene; and aliphatichydrocarbons such as n-pentane, n-hexane, n-heptane, and cyclohexane. Inparticular, the combination of one or more of alcohols such as methanol,ethanol, n-propyl alcohol, isopropyl alcohol, n-butanol, andtert-butanol; and one or more of aromatic hydrocarbons such as benzene,toluene, xylene, chlorobenzene, and trifluoromethylbenzene; is morepreferred since both effective purification and high recovery can beachieved. Considering ease of removal of the solvent by drying afterfiltration, and safety of the operator, the combination of methanol,ethanol, or isopropyl alcohol; and toluene; is most preferred.

[Method for Producing α-Substituted Cysteine Represented by GeneralFormula (1S) or Salt Thereof]

Among the α-substituted cysteines represented by the General Formula (1)and salts thereof, α-substituted-D-cysteines having an(S)-configuration, represented by General Formula (1S):

(wherein R² represents a C₁-C₄ alkyl group),and salts thereof are useful as intermediates for pharmaceuticals andthe like. In particular, α-methyl-D-cysteine and salts thereof, in whichR² is methyl, are known to be useful as intermediates for hyperferremia,and establishment of a production method which enables theirindustrial-scale production has been expected.

Specific examples of the α-substituted cysteine represented by theGeneral Formula (1S) include the following compounds.

Among the compounds mentioned above, the following compound is preferredas the compound (1S).

In cases where an α-substituted-D-cysteine represented by GeneralFormula (15) or a salt thereof is used as an intermediate for apharmaceutical, high optical purity is required as described above. Ingeneral, the optical purity is preferably not less than 99.0% e.e., morepreferably not less than 99.5% e.e., especially preferably not less than99.8% e.e.

The method for producing an α-substituted-D-cysteine represented byGeneral Formula (1S) or a salt thereof is described below. The reactionconditions in each step are the same as those described in [Method forProducing α-Substituted Cysteine Represented by General Formula (1) orSalt Thereof].

In order to produce an α-substituted-D-cysteine represented by GeneralFormula (1S) or a salt thereof, the steps (ii) to (v) described abovemay be carried out using the compound represented by General Formula(3S):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted; and R² has the samemeaning as in the General Formula (1S)) produced in the step (i)described above.

First, in the step (ii), a condensing agent or an acid halogenatingagent may be allowed to act on the General Formula (3S), to obtain acompound represented by General Formula (4S):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (3S); and X represents —OP(O)(OPh)₂, —OP(O)(OEt)₂, —OC(O)OR³, ora halogen atom).The compound represented by General Formula (4S) may be either an(S)-isomer or an (R)-isomer, depending on the type of X.

In particular, it is preferred to allow a chloroformic ester to act toobtain a compound represented by General Formula (4S-1):

(wherein R¹, R², and R³ have the same meanings as R¹, R², and R³ in theGeneral Formula (4)), in which X is —OC(O)OR³.

Specific examples of the compound represented by the General Formula(4S-1) include the following compounds.

Among the compounds mentioned above, the following compounds arepreferred as the compound (4S-1).

Subsequently, in the step (iii), an azidation agent may be allowed toact on the compound represented by General Formula (4S), to obtain acompound represented by General Formula (5S):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (4S)).

In particular, it is preferred to use, as a material, the compoundrepresented by the General Formula (4S-1), in which X is —OC(O)OR³.

Specific examples of the compound represented by the General Formula(5S) include the following compounds.

Among the compounds mentioned above, the following compounds arepreferred as the compound represented by the General Formula (5S).

Subsequently, in the step (iv), the compound represented by the GeneralFormula (5S) may be converted to a compound represented by GeneralFormula (6S):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (5S))by Curtius rearrangement reaction.

Specific examples of the compound represented by the General Formula(6S) include the following compounds.

Among the compounds mentioned above, the following compounds arepreferred as the compound represented by the General Formula (6S).

Subsequently, in the step (v), the compound of General Formula (1S) isobtained through the processes of: (a) converting the isocyanate groupto an amino group; (b) hydrolyzing the ester group to allow itsconversion to a carboxyl group; and (c) allowing an acid to act forremoval of the tert-butyl group. In particular, after obtaining thecompound represented by the General Formula (6S), the reaction ispreferably allowed to proceed through the steps (vi-1), (vi-2), and(vii) described above.

That is, in the step (vi-1), an acid is allowed to act on the GeneralFormula (6S) to construct a thiazolidinone ring, thereby allowingconversion to a compound represented by General Formula (7S-1):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (6S)).

Subsequently, in the step (vi-2), an acid or a base is allowed to act onthe compound represented by the General Formula (7S-1) to hydrolyze theester group, thereby allowing conversion to a compound represented byGeneral Formula (7S-2):

(wherein R² has the same meaning as R² in the General Formula (7S-1)).Subsequently, in the step (vii), an acid or a base is allowed to act onthe General Formula (7S-2) to open the thiazolidinone ring, therebyobtaining the α-substituted-D-cysteine represented by the GeneralFormula (1S) or the salt thereof.

In other words, by allowing the reaction to proceed through the step(vi-1), elimination of the tert-butyl group, which generally requires along time, can be allowed to proceed quickly, and the compoundrepresented by General Formula (7S-1) can be obtained in a short time.Since the compound represented by General Formula (7S-2) obtained in thesubsequent step (vi-2) has high crystallinity, and low solubility inwater at a pH of not more than the point of neutralization and innonpolar solvents, both water-soluble impurities including inorganicsalts and water-insoluble impurities including reaction intermediatescan be easily removed by crystallization. Therefore, after thesubsequent step (vii), the α-substituted-D-cysteine represented byGeneral Formula (1S) or the salt thereof can be obtained with highpurity. Moreover, since the compound represented by General Formula(7S-2) has higher crystallinity than the racemic body of the compound,its optical purity can be easily increased by crystallization.Therefore, the compound represented by the General Formula (7S) below:

(wherein R² has the same meaning as in General Formula (6); and R⁵represents a hydrogen atom, or has the same meaning as R¹ in the GeneralFormula (6S)) can be said to be an important intermediate for producinga high-quality α-substituted cysteine represented by General Formula(1S) or a salt thereof in a short time.

Here, General Formula (7S) includes General Formula (7S-1) and GeneralFormula (7S-2).

Specific examples of the compound represented by the General Formula(7S) include the following compounds.

Among the compounds mentioned above, the following compounds arepreferred as the compound represented by General Formula (7S).

In cases where an α-substituted-D-cysteine represented by GeneralFormula (1S) or a salt thereof is used as an intermediate for apharmaceutical, high optical purity is required as described above.Therefore, in general, the optical purity of the compound represented byGeneral Formula (7S), which is the precursor, is also preferably notless than 99.0% e.e., more preferably not less than 99.5% e.e.,especially preferably not less than 99.8% e.e. In cases where theoptical purity of General Formula (7-2) does not reach a desired levelafter carrying the step (vi-2), recrystallization may be repeated toincrease the optical purity.

[Method for Producing Compound Represented by General Formula (2)]

The starting substance for the steps (i) to (vii) described above is acompound represented by General Formula (2):

(wherein each R¹ independently represents a C₁-C₁₀ alkyl group which isoptionally substituted, a C₇-C₂₀ aralkyl group which is optionallysubstituted, or a C₆-C₁₂ aryl group which is optionally substituted, andR² represents a C₁-C₄ alkyl group). Conventional methods for producingthis compound have been industrially unfavorable since bischloromethylether, which is a carcinogenic substance, may be generated.

In view of this, the present inventors discovered a method for safelyproducing the compound represented by General Formula (2) withoutgeneration of bischloromethyl ether. The method is described below.

The method for producing the compound represented by General Formula (2)comprises either the steps (viii) to (x) or the step (xi).

<Step (viii)>

First, the step (viii) is described below.

The step (viii) is a step of reacting tert-butyl mercaptan withformaldehyde to produce tert-butylthiomethanol.

As the tert-butyl mercaptan, a commercially available product may beused. As the formaldehyde, any of commercially available products in theform of paraformaldehyde, formalin, or 1,3,5-trioxane may be used. Fromthe viewpoint of the cost and ease of handling, formalin is preferred.

The amount of formaldehyde may be appropriately set within the range of0.7 molar equivalent to 10 molar equivalents with respect to the amountof tert-butyl mercaptan. In order to quantitatively obtaintert-butylthiomethanol, the amount is preferably 0.9 molar equivalent to2.0 molar equivalents. From the viewpoint of the cost and ease ofpost-treatment, the amount is more preferably 0.9 molar equivalent to1.2 molar equivalents.

In the this step, a solvent may be used. However, since the reactionproceeds even in cases where the formalin is used as a solvent withoutusing another solvent, it is preferred not to use another solvent, fromthe viewpoint of the cost.

The reaction temperature may be appropriately set within the range of,for example, −50° C. to 200° C. The reaction temperature is preferably−20° C. to 100° C. Considering reactivity and the boiling point oftert-butyl mercaptan, the reaction temperature is more preferably 40° C.to 80° C.

The reaction time may be appropriately set within the range of, forexample, 0.5 hour to 100 hours. The reaction time is preferably 1 hourto 40 hours, more preferably 5 hours to 20 hours.

In cases where formaldehyde remains in the tert-butylthiomethanol afterthe reaction of the this step, the formaldehyde may react with asubstance that generates hydrogen chloride such as a chlorination agentin the step (ix) described below, to cause generation of bischloromethylether, which is a carcinogenic substance. Thus, in cases where excessiveformaldehyde is used, a step for removing the formaldehyde needs to beincluded as a post-treatment. For example, a method in which water and awater-insoluble organic solvent are added to perform washing with waterfor removing formaldehyde into the aqueous layer, a method in whichtert-butylthiomethanol is purified by distillation, a method in whichsolid paraformaldehyde is removed by filtration, or the like may beemployed. Among these, the method in which water and a water-insolubleorganic solvent are added to perform washing with water is preferredsince formaldehyde can be efficiently removed by the method.

The water-insoluble organic solvent to be used for the washing withwater include aromatic hydrocarbons such as benzene, toluene, xylene,and chlorobenzene; aliphatic hydrocarbons such as n-pentane, n-hexane,n-heptane, and cyclohexane; esters such as methyl acetate, ethylacetate, and isopropyl acetate; ethers such as diethyl ether,di-n-propyl ether, di-n-butyl ether, methyl isopropyl ether, andcyclopentyl methyl ether; and halogenated hydrocarbons such asdichloromethane, chloroform, carbon tetrachloride, and1,2-dichloroethane. Among these, aliphatic hydrocarbons such asn-pentane, n-hexane, n-heptane, and cyclohexane are preferred sincethese are highly separable from water, can be easily removed bydistillation because of their low boiling points, and can be reused.

Depending on the water-insoluble organic solvent used, and in caseswhere water is used as a solvent, water in which an inorganic salt isdissolved is preferably used in order to minimize loss oftert-butylthiomethanol into the aqueous layer during the washing withwater. From the viewpoint of the cost, saline is more preferably used.

<Step (ix)>

The step (ix) is described below.

The step (ix) is a step of allowing a chlorination agent to act, in thepresence of a base, on the tert-butylthiomethanol obtained in the step(viii), to obtain tert-butylthiochloromethane.

Examples of the chlorination agent include hydrogen chloride, pivaloylchloride, acetyl chloride, benzoyl chloride, thionyl chloride,phosphorous trichloride, phosphorous pentachloride, phosphorousoxytrichloride, oxalyl chloride, and sulfuryl chloride. Among these,thionyl chloride is preferred since it is inexpensive and shows highreaction selectivity.

The amount of the chlorination agent used may be appropriately setwithin the range of, for example, 0.7 molar equivalent to 10 molarequivalents with respect to the amount of tert-butylthiomethanol. Fromthe viewpoint of the cost and ease of post-treatment, the amount is morepreferably 0.8 molar equivalent to 3 molar equivalents. In order toquantitatively obtain tert-butylthiochloromethane, the amount is morepreferably 0.9 molar equivalent to 1.2 molar equivalents.

The base is not limited as long as the base does not havenucleophilicity, and examples of the base include pyridines such aspyridine, 2-chloropyridine, 3-chloropyridine, 2-methylpyridine,3-methylpyridine, 2,3-lutidine, 2,4-lutidine, 2,5-lutidine,2,6-lutidine, 3,4-lutidine, and 3,5-lutidine; and tertiary amines suchas triethylamine, triisopropylamine, dimethylisopropylamine,dimethylbutylamine, diethylmethylamine, diisopropylethylamine, andN-methylmorpholine. From the viewpoint of the cost, pyridine,triethylamine, and diisopropylethylamine are preferred. Pyridine is morepreferred from the viewpoints of the fact that the solubility of thehydrochloric acid salt produced as a by-product in the later-describedpost-treatment method is advantageously low, and of the yield.

The amount of the base used may be appropriately set within the rangeof, for example, 0.7 molar equivalent to 10 molar equivalents withrespect to the amount of tert-butylthiomethanol. The amount ispreferably 0.8 molar equivalent to 5 molar equivalents. From theviewpoint of the cost and ease of post-treatment, the amount is morepreferably 0.9 molar equivalent to 1.5 molar equivalents. The amount ismost preferably 1.0 molar equivalent to 1.1 molar equivalents withrespect to the amount of the chlorination agent used.

In the step (ix), a reaction solvent may be used. Examples of thereaction solvent include ketones such as acetone, 2-butanone, andmethylisobutyl ketone; aromatic hydrocarbons such as benzene, toluene,xylene, chlorobenzene, and trifluoromethylbenzene; aliphatichydrocarbons such as n-pentane, n-hexane, n-heptane, and cyclohexane;esters such as methyl acetate, ethyl acetate, and isopropyl acetate;ethers such as diethyl ether, di-n-propyl ether, di-n-butyl ether,methyl isopropyl ether, methyl-tert-butyl ether, ethyl-tert-butyl ether,cyclopentyl methyl ether, tetrahydrofuran, dioxane, and1,2-dimethoxyethane; amides such as N,N-dimethylformamide andN,N-dimethylacetamide; nitriles such as acetonitrile and propionitrile;halogenated hydrocarbons such as dichloromethane, chloroform, carbontetrachloride, and 1,2-dichloroethane; and dimethylsulfoxide.

Among these, toluene, xylene, chlorobenzene, n-pentane, n-hexane,n-heptane, cyclohexane, dichloromethane, and chloroform are preferredsince these are inactive against acids, hardly cause side reactions, andcan be reused. Aliphatic hydrocarbons such as n-pentane, n-hexane,n-heptane, and cyclohexane are more preferred since these are solventsin which the hydrochloric acid salt of the base produced as a by-productis hardly dissolved and can therefore be easily removed in thelater-described post-treatment process.

The amount of water and alcohols contained is preferably minimum sincethey easily cause degradation of the chlorination agent.

The amount of the reaction solvent used may be appropriately set withinthe range of, for example, 0 volume to 100 volumes with respect to theamount of tert-butylthiomethanol. In cases where the solvent is notused, there is a problem in the safety of the process since a largeamount of heat is generated in this step. From this view point, and alsofrom the viewpoint of the volume efficiency, it is preferred to use 1volume to 20 volumes of the reaction solution with respect to the amountof tert-butylthiomethanol. Considering securing of the stirringefficiency upon precipitation of the hydrochloric acid salt of the baseas a by-product, and the volume efficiency, the amount of the reactionsolvent is more preferably 5 volumes to 15 volumes.

The reaction temperature may be appropriately set within the range of,for example, −80° C. to 100° C. The reaction temperature is preferably−50° C. to 50° C. Since tert-butylthiochloromethane is degraded to causea decrease in the yield in cases where the temperature is too high, thereaction temperature is more preferably −15° C. to 25° C.

The reaction time may be appropriately set within the range of, forexample, 0.5 hour to 50 hours. The reaction time is preferably 1 hour to20 hours. Since, in cases where the reaction is carried out for a longtime, degradation of tert-butylthiochloromethane occurs, leading to adecrease in the yield, the reaction time is more preferably 1 hour to 8hours.

As a post-treatment after this process, the hydrochloric acid salt ofthe base precipitated in the reaction solution may be removed byfiltration or washing with water. After the removal, the solvent may besimply removed depending on the purity, or purification by distillationmay be carried out. For example, in cases where pyridine is used as thebase, simple removal of the solvent after filtration is sufficient sincethe solubility of pyridine hydrochloride in the solvent is low asdescribed above. However, in addition, the operation of distillation ispreferably carried out in order to obtain highly puretert-butylthiochloromethane.

<Step (x)>

The step (x) is described below.

The step (x) is a step of allowing a compound represented by GeneralFormula (8):

(wherein each R¹ independently represents a C₁-C₁₀ alkyl group which isoptionally substituted, a C₇-C₂₀ aralkyl group which is optionallysubstituted, or a C₆-C₁₂ aryl group which is optionally substituted, andR² represents a C₁-C₄ alkyl group) to act, in the presence of a base, onthe tert-butylthiochloromethane obtained in the step (ix), to obtain acompound represented by General Formula (2):

(wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (8)).

The R¹ and the R² in the compound represented by General Formula (8) arethe same as those described in the step (i).

Thus, specific examples of the compound represented by General Formula(8) include the following compounds.

Among the compounds mentioned above, the following compounds arepreferred as the compound represented by the General Formula (8).

As the compound represented by General Formula (8), a commerciallyavailable product may be used. Alternatively, the compound representedby General Formula (8) may be produced according to, for example, themethod described in Tetrahedron Letters (2008), 49, (15), 2446-2449,using the combination of a dialkyl malonate, a base, and an alkylatingagent

In the compound represented by General Formula (8), the R¹s are notnecessarily the same, and may be different from each other. In caseswhere the R¹s are different from each other, two enantiomers, the(R)-isomer and the (S)-isomer, are present as the compound representedby the General Formula (8). The mixing ratio between these enantiomersis not limited.

The amount of tert-butylthiochloromethane used may be appropriately setwithin the range of, for example, 0.7 molar equivalent to 10 molarequivalents with respect to the amount of the compound represented bythe General Formula (8). In order to quantitatively obtain the compoundrepresented by General Formula (2), the amount is preferably 0.9 molarequivalent to 5 molar equivalents. The amount is more preferably 1.0molar equivalent to 1.5 molar equivalents from the viewpoint of thecost.

Examples of the base include alkali metal alkoxides and alkali metalhydrides. Examples of the alkali metal alkoxides include lithiumethoxide, lithium methoxide, lithium tert-butoxide, sodium ethoxide,sodium methoxide, sodium tert-butoxide, potassium ethoxide, potassiummethoxide, and potassium tert-butoxide. Examples of the alkali metalhydrides include lithium hydride, sodium hydride, and potassium hydride.

Among these, alkali metal alkoxides such as sodium ethoxide, sodiummethoxide, sodium tert-butoxide, potassium ethoxide, potassiummethoxide, and potassium tert-butoxide are preferred since they do notgenerate hydrogen during the reaction and can be safely handled, and theoperation of removing mineral oil, which is contained in alkali metalhydrides, is not required. Potassium tert-butoxide is more preferredsince it is highly soluble in organic solvents and has lownucleophilicity.

The amount of the base may be appropriately set within the range of, forexample, 0.7 molar equivalent to 10 molar equivalents with respect tothe amount of the compound represented by General Formula (8). In orderto quantitatively obtain the compound represented by General Formula(2), the amount is preferably 0.9 molar equivalent to 5 molarequivalents. From the viewpoint of the cost and reduction of sidereactions, the amount is more preferably 0.9 molar equivalent to 1.5molar equivalents.

In this step, a reaction solvent may be used. Examples of the reactionsolvent include aromatic hydrocarbons such as benzene, toluene, xylene,chlorobenzene, and trifluoromethylbenzene; aliphatic hydrocarbons suchas n-pentane, n-hexane, n-heptane, and cyclohexane; ethers such asdiethyl ether, di-n-propyl ether, di-n-butyl ether, methyl isopropylether, methyl-tert-butyl ether, ethyl-tert-butyl ether, cyclopentylmethyl ether, tetrahydrofuran, dioxane, and 1,2-dimethoxyethane; amidessuch as N,N-dimethylformamide and N,N-dimethylacetamide; halogenatedhydrocarbons such as dichloromethane, chloroform, carbon tetrachloride,and 1,2-dichloroethane; and dimethylsulfoxide.

Among these, aprotic polar solvents such as tetrahydrofuran, dioxane,1,2-dimethoxyethane, N,N-dimethylformamide, N,N-dimethylacetamide, anddimethylsulfoxide are preferred since the alkaline metal salt producedby the reaction between the compound represented by General Formula (8)and the base is highly soluble in these solvents, and the solventstherefore have an effect to promote the reaction. Tetrahydrofuran ismore preferred since it is inexpensive and easily available.

The amount of the reaction solvent used may be appropriately set withinthe range of, for example, 0 volume to 100 volumes with respect to theamount of tert-butylthiochloromethane. In particular, from the viewpointof the fact that the reaction more easily proceeds in cases where asolvent in which the alkaline metal salt of the compound represented byGeneral Formula (8) dissolves is added, and from the viewpoint of thevolume efficiency, the amount of the reaction solvent is preferably 1volume to 20 volumes, more preferably 3 volumes to 8 volumes.

The reaction temperature may be appropriately set within the range of,for example, −100° C. to 200° C. The reaction temperature is preferably−50° C. to 100° C. In cases where the temperature is too low, solubilityof the alkaline metal salt of the compound represented by GeneralFormula (8) in the solvent decreases, so that the reaction proceedsslowly, while in cases where the temperature is too high, a sidereaction may occur, leading to a decrease in the yield. Thus, thereaction temperature is more preferably −10° C. to 50° C.

The reaction time may be appropriately set within the range of, forexample, 0.5 hour to 100 hours. The reaction time is preferably 1 hourto 20 hours. In cases where the reaction time is too long, a sidereaction may occur, leading to a decrease in the yield. Thus, thereaction time is more preferably 1 hour to 10 hours.

Preferably, as a post-treatment after this reaction, the inorganic saltprecipitated in the reaction solution may be removed by filtration, orwater and a water-insoluble solvent may be added to the solution toremove the inorganic salt by washing with water. In particular, since,depending on the reaction solvent used, the inorganic salt is dissolvedin the reaction solvent, washing water is more preferably included. Whenwater is added to the reaction solution, the aqueous layer becomesbasic. In order to suppress hydrolysis of the ester group in thecompound represented by General Formula (8), a method in which acidicwater is added to the reaction solution at low temperature, a method inwhich an acid is added after the addition of water, to adjust the pH ofthe aqueous layer to a neutral pH, a method in which the reactionsolution is added to an acidic aqueous solution, or the like may beemployed. It is preferred to add an acid after the addition of water toadjust the pH of the aqueous layer to a neutral pH. After the removal ofthe inorganic salt, depending on the purity, the solvent may be simplyremoved by concentration, or purification on a column may be carriedout.

Examples of the water-insoluble solvent used for the extraction in thepost-treatment include ketones such as 2-butanone, methylisobutylketone, and cyclohexanone; aromatic hydrocarbons such as benzene,toluene, xylene, chlorobenzene, and trifluoromethylbenzene; aliphatichydrocarbons such as n-pentane, n-hexane, n-heptane, and cyclohexane;esters such as methyl acetate, ethyl acetate, isopropyl acetate, andbutyl acetate; ethers such as diethyl ether, di-n-propyl ether,diisopropyl ether, di-n-butyl ether, methyl isopropyl ether,methyl-tert-butyl ether, ethyl-tert-butyl ether, and cyclopentyl methylether; and halogenated hydrocarbons such as dichloromethane, chloroform,carbon tetrachloride, and 1,2-dichloroethane.

Among these, toluene, xylene, diisopropyl ether, di-n-butyl ether,methyl isopropyl ether, and methyl-tert-butyl ether are preferred sincethe compound represented by General Formula (2) shows high solubility inthese solvents. From the viewpoint of the cost, toluene is morepreferred.

By the steps (viii) to (x), the compound represented by General Formula(2), which is the starting substance, can be produced.

By the step (xi) described below, the compound represented by GeneralFormula (2) can be produced by a method different from (viii) to (x).

<Step (xi)>

The step (xi) is a step of allowing an alkylating agent to act on acompound represented by General Formula (9):

(wherein each R¹ independently represents a C₁-C₁₀ alkyl group which isoptionally substituted, a C₇-C₂₀ aralkyl group which is optionallysubstituted, or a C₆-C₁₂ aryl group which is optionally substituted)in the presence of a base, to obtain a compound represented by GeneralFormula (2):

(wherein R¹ has the same meaning as R¹ in the General Formula (9), andR² represents a C₁-C₄ alkyl group).

The compound represented by General Formula (9) can be obtained by, forexample, allowing tert-butyl mercaptan to act on dialkyl methylenemalonate synthesized by the method described in WO 2010/129066 orOrganic synthesis, 1958, 38, 22.

The R¹ and the R² in the compound represented by the General Formula (9)are the same as those described in the step (i).

Thus, specific examples of the compound represented by General Formula(9) include the following compounds.

Among the compounds mentioned above, the following compounds arepreferred as the compound represented by the General Formula (9).

In the compound represented by General Formula (9), the R¹s are notnecessarily the same, and may be different from each other. In caseswhere the R¹s are different from each other, two enantiomers, the(R)-isomer and the (S)-isomer, are present as the compound representedby the General Formula (9). The mixing ratio between these enantiomersis not limited.

Examples of the alkylating agent which may be used include R²Y and(R²O)₂SO₂ (wherein R² has the same meaning as in the General Formula(2); and Y represents a halogen atom, or a C₆-C₁₂ arylsulfonyloxy group,C₁-C₁₀ alkylsulfonyloxy group, or C₇-C₂₀ aralkylsulfonyloxy group, whichis optionally substituted).

Examples of the halogen atom of Y include a chlorine atom, bromine atom,and iodine atom. Examples of the C₆-C₁₂ arylsulfonyloxy group includebenzenesulfonyloxy, 1-naphthalenesulfonyloxy, and2-naphthalenesulfonyloxy. Examples of the C₁-C₁₀ alkylsulfonyloxy groupinclude methanesulfonyloxy, ethanesulfonyloxy, and propanesulfonyloxy.Examples of the C₇-C₂₀ aralkylsulfonyloxy group includebenzylsulfonyloxy. Examples of substituents which may be contained inthe arylsulfonyloxy group, alkylsulfonyloxy group, or aralkylsulfonyloxygroup include C₁-C₆ alkyl groups such as methyl and ethyl; C₁-C₆ alkoxygroups such as methoxy and ethoxy; halogen atoms such as a fluorineatom, chlorine atom, bromine atom, and iodine atom; and nitro. Thenumber of substituents is not limited, and, in cases where there are twoor more substituents, the substituents may be of the same type ordifferent types.

As R², a chlorine atom, bromine atom, iodine atom, methanesulfonyloxy,trifluoromethanesulfonyloxy, or 4-toluenesulfonyloxy is preferred. Amongthese, a bromine atom, iodine atom, methanesulfonyloxy, orp-toluenesulfonyloxy is preferred because of their high reactivity. Abromine atom is more preferred.

The amount of the alkylating agent used may be appropriately set withinthe range of, for example, 0.7 molar equivalent to 10 molar equivalentswith respect to the amount of the compound represented by GeneralFormula (9). In order to allow the reaction to proceed quickly and tothereby suppress side reactions, the amount of the agent is preferablyslightly excessive. Thus, the amount is preferably 1.0 molar equivalentto 3.0 molar equivalents. From the viewpoints of the cost, and of thefact that side reactions easily proceed in cases where the amount of theagent is too excessive, the amount of the agent is more preferably 1.1molar equivalents to 2.0 molar equivalents.

Examples of the base include alkali metal alkoxides and alkali metalhydrides. Examples of the alkali metal alkoxides include lithiumethoxide, lithium methoxide, lithium tert-butoxide, sodium ethoxide,sodium methoxide, sodium tert-butoxide, potassium ethoxide, potassiummethoxide, and potassium tert-butoxide. Examples of the alkali metalhydrides include lithium hydride, sodium hydride, and potassium hydride.

The compound represented by General Formula (9) repeats elimination andaddition of a tert-butylthio group in the presence of a base. Occurrenceof the elimination results in conversion of the General Formula (9) todialkyl methylene malonate. In cases where a nucleophilic base ispresent in this process, Michael addition reaction to the dialkylmethylene malonate may occur, leading to a decrease in the yield. On theother hand, bases having no nucleophilicity do not cause such a sidereaction. Thus, the base is preferably an alkali metal hydride such aslithium hydride, sodium hydride, or potassium hydride, which does nothave nucleophilicity. The base is more preferably sodium hydride fromthe viewpoint of the cost and safety.

The amount of the base used may be appropriately set within the rangeof, for example, 0.7 molar equivalent to 10 molar equivalents withrespect to the amount of the compound represented by General Formula(9). Since partial inactivation tends to occur due to water in thesolvent or the like, the amount is preferably 0.9 molar equivalent to4.0 molar equivalents. From the viewpoint of the cost and suppression ofside reactions, the amount is more preferably 1.0 molar equivalent to2.0 molar equivalents.

Examples of the reaction solvent include aromatic hydrocarbons such asbenzene, toluene, xylene, chlorobenzene, and trifluoromethylbenzene;aliphatic hydrocarbons such as n-pentane, n-hexane, n-heptane, andcyclohexane; ethers such as diethyl ether, di-n-propyl ether, di-n-butylether, methyl isopropyl ether, methyl-tert-butyl ether, ethyl-tert-butylether, cyclopentyl methyl ether, tetrahydrofuran, dioxane, and1,2-dimethoxyethane; amides such as N,N-dimethylformamide andN,N-dimethylacetamide; halogenated hydrocarbons such as dichloromethane,chloroform, carbon tetrachloride, and 1,2-dichloroethane; anddimethylsulfoxide.

Among these, aprotic polar solvents such tetrahydrofuran, dioxane,1,2-dimethoxyethane, N,N-dimethylformamide, N,N-dimethylacetamide, anddimethylsulfoxide are preferred since the salt produced by the reactionbetween the compound represented by General Formula (9) and the base ishighly soluble in these solvents, and the solvents therefore have aneffect to promote the reaction. Tetrahydrofuran is more preferred sinceit has a low boiling point and can be easily removed by concentration.

The amount of the reaction solvent used may be appropriately set withinthe range of, for example, 0 volume to 100 volumes with respect to theamount of the compound represented by General Formula (9). Depending onthe type of the base, the amount of heat of the reaction is large, andit is risky to carry out the reaction without suspension in a solvent.Because of this, and from the viewpoint of the volume efficiency, theamount of the reaction solvent is preferably 1 volume to 30 volumes,more preferably 8 volumes to 20 volumes.

The reaction temperature may be appropriately set within the range of,for example, −100° C. to 200° C. The reaction temperature is preferably−50° C. to 100° C. In cases where the reaction temperature is too low,the solubility of the salt composed of the compound represented byGeneral Formula (9) and the base in the reaction solvent decreases,leading to a decrease in the reaction rate, while in cases where thereaction temperature is too high, side reactions such as generation of adimer may occur. Therefore, the reaction temperature is more preferably−10° C. to 50° C.

The reaction time may be appropriately set within the range of, forexample, 0 hour to 30 hours. The reaction time is preferably 5 minutesto 5 hours. Since, in case where the reaction time is too long, sidereactions may occur, the reaction time is more preferably 15 minutes to2 hours.

As a post-treatment after this reaction, the inorganic salt precipitatedin the reaction solution is preferably removed by filtration or washingwith water. Since, depending on the solvent used, the inorganic salt isdissolved in the reaction solvent, washing water is more preferablyincluded. When water is added to the reaction solution, the aqueouslayer becomes basic. In order to suppress hydrolysis of the ester groupin the compound represented by General Formula (8) or General Formula(9), a method in which acidic water is added to the reaction solution atlow temperature, a method in which an acid is added after the additionof water, to adjust the pH of the aqueous layer to a neutral pH, amethod in which the reaction solution is added to an acidic aqueoussolution, or the like may be employed. It is preferred to add an acidafter the addition of water, to adjust the pH of the aqueous layer to aneutral pH. After the removal of the inorganic salt, depending on thepurity, the solvent may be simply removed by concentration, orpurification on a column may be carried out.

In the washing with water in the post-treatment of this step, examplesof the water-insoluble solvent which may be used for the extractioninclude ketones such as 2-butanone, methylisobutyl ketone, andcyclohexanone; aromatic hydrocarbons such as benzene, toluene, xylene,chlorobenzene, and trifluoromethylbenzene; aliphatic hydrocarbons suchas n-pentane, n-hexane, n-heptane, and cyclohexane; esters such asmethyl acetate, ethyl acetate, isopropyl acetate, and butyl acetate;ethers such as diethyl ether, di-n-propyl ether, diisopropyl ether,di-n-butyl ether, methyl isopropyl ether, methyl-tert-butyl ether,ethyl-tert-butyl ether, and cyclopentyl methyl ether; and halogenatedhydrocarbons such as dichloromethane, chloroform, carbon tetrachloride,and 1,2-dichloroethane.

Among these, n-pentane, n-hexane, n-heptane, cyclohexane, diethyl ether,di-n-propyl ether, diisopropyl ether, di-n-butyl ether, methyl isopropylether, methyl-tert-butyl ether, ethyl-tert-butyl ether, anddichloromethane are preferred since these have low boiling points, andshow high removal efficiencies. Diethyl ether, di-n-propyl ether,diisopropyl ether, di-n-butyl ether, methyl isopropyl ether,methyl-tert-butyl ether, and ethyl-tert-butyl ether are more preferredsince they show high extraction efficiencies.

[Novel Compounds]

By the present invention, the following novel compounds can be provided.

The novel compounds of the present invention can be produced by theproduction methods of the present invention, and can also be synthesizedusing ordinary techniques of organic chemistry.

Specific examples of R¹, R², and R³ in the following General Formulae(4-1), (5), (6), and (7S), and specific examples of the compoundsrepresented by General Formulae (4-1), (5), (6), and (7S) are the sameas those described in the [Method for Producing α-Substituted CysteineRepresented by General Formula (1) or Salt Thereof] and the [Method forProducing α-Substituted Cysteine Represented by General Formula (1S) orSalt Thereof] mentioned above.

<Compound Represented by General Formula (4-1)>

A compound represented by General Formula (4-1):

(wherein R¹ and R³ each independently represent a C₁-C₁₀ alkyl groupwhich is optionally substituted, a C₇-C₂₀ aralkyl group which isoptionally substituted, or a C₆-C₁₂ aryl group which is optionallysubstituted, and R² represents a C₁-C₄ alkyl group).

<Compound Represented by General Formula (5)>

A compound represented by General Formula (5):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² represents aC₁-C₄ alkyl group).

<Compound Represented by General Formula (6)>

A compound represented by General Formula (6):

(wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² represents aC₁-C₄ alkyl group).

<Compound Represented by General Formula (7S)>

A compound represented by General Formula (7S):

(wherein R² represents a C₁-C₄ alkyl group, and R⁵ represents a hydrogenatom, or a C₁-C₁₀ alkyl group which is optionally substituted, a C₇-C₂₀aralkyl group which is optionally substituted, or a C₆-C₁₂ aryl groupwhich is optionally substituted).

EXAMPLES

The present invention is described in more detail by way of Examples.However, the present invention is not limited thereto.

In the present Examples, quantitative analysis was carried out by GC(Gas Chromatography) or HPLC (High Performance Liquid Chromatography)under the following conditions.

<GC-1>

Column: GL Science TC-5 (0.53 mm×300 mm; film thickness, 5 μm)

Carrier gas: helium; column flow, 3.93 mL/minute (split ratio, 20:1);inlet pressure, 22.2 kPa; total flow, 85.4 mL/minute (linear velocity,30 cm/second)

Column Temperature: 50° C. (kept for 5 minutes)→heating at 10°C./minute→200° C. (kept for 10 minutes)

Injection temperature: 220° C.

Detection temperature: 230° C.

Detector: FID; hydrogen, 40 mL/minute; air, 400 mL/minute; make-up gas,nitrogen, 30 mL/minute; signal attenuation, ×2-3

Sample injection volume: 1.0 μL

<HPLC-1>

Column: Nacalai Tesque, COSMOSIL 5C18-MSII (4.6 mm×250 mm, particlesize, 5 μm)

Mobile phase: 50 mmol/L aqueous tetrafluoroacetic acidsolution/acetonitrile=30/70 (volume ratio)

Flow rate: 1.0 mL/minute

Column temperature: 35° C.

Detection wavelength: UV 220 nm

<HPLC-2>

Column: DAICEL, CHIRALPAK OZ-3R (4.6 mm×150 mm)

Mobile phase: 0.02 wt % aqueous tetrafluoroacetic acidsolution/acetonitrile=75/25 (volume ratio)

Flow rate: 0.7 mL/minute

Column temperature: 30° C.

Detection wavelength: UV 220 nm

<HPLC-3>

Column: SIELC, primesep 100 (4.6 mm×150 mm, particle size, 5 μm)

Mobile phase: A, 0.1 wt % aqueous tetrafluoroacetic acid solution; B,acetonitrile

Gradient (B concentration): 10% at Minute 0→30% at Minute 15→80% atMinute 20

Flow rate: 1.0 mL/minute

Column temperature: 40° C.

Detection wavelength: UV 210 nm

<HPLC-4>

Column: Shiseido, Chiral CD-Ph (4.6 mm×250 mm, particle size, 5 μm)

Mobile phase: aqueous (100 mmol/L perchloric acid+0.2 wt % phosphoricacid) solution/acetonitrile=99/1 (volume ratio)

Flow rate: 0.5 mL/minute

Column temperature: 30° C.

Detection wavelength: UV 205 nm

[Example 1] Step (viii): Production of tert-Butylthiomethanol

Under nitrogen gas flow, 240 g (2.66 mol) of tert-butyl mercaptan and216 g (2.66 mol) of formalin (37 wt %) were placed in a 1-L reactor atroom temperature, and the resulting mixture was stirred. After allowingthe reaction to proceed at 60° C. for 9 hours, separation extraction wascarried out by adding 160 g of cyclohexane while keeping the reactor ata temperature within the range of 50° C. to 60° C., followed by removingthe aqueous phase. Thereafter, cyclohexane was removed by concentratingthe cyclohexane layer under reduced pressure. The resulting crudetert-butylthiomethanol was subjected to distillation under reducedpressure at 70° C./2.0 kPa to obtain 208 g (1.73 mmol; yield, 65.0%) oftert-butylthiomethanol. As a result of GC analysis under <GC-1>, itspurity was found to be 99% by area. The measurement results obtainedwere as follows.

¹H-NMR (400 MHz, CDCl₃) δ 1.41 (9H, s), 2.11 (¹H, t, J=6.4 Hz), 4.83(2H, d, J=6.4 Hz).

[Example 2] Step (ix): Production of tert-Butylthiochloromethane

Under nitrogen gas flow, 190 g (1.58 mol) of tert-butylthiomethanolproduced in Example 1 and 1.44 kg of cyclohexane were placed in a 3-Lreactor at room temperature. To the resulting mixture, 150 g (1.90 mol)of pyridine was added while the inner temperature was kept at 0° C. Tothe mixture, 226 g (1.89 mol) of thionyl chloride was then addeddropwise while the inner temperature was kept at −2° C. to 6° C. Afterstirring the resulting mixture at a temperature within the range of −4°C. to 0° C. for 1 hour, 380 g of cyclohexane was added thereto, and theprecipitated salt was removed by filtration. Cyclohexane was removed byconcentrating the obtained filtrate by distillation, and the resultingcrude tert-butylthiochloromethane was subjected to distillation underreduced pressure at 57° C./3.0 kPa to obtain 140 g (1.01 mol; yield,63.9%) of tert-butylthiochloromethane. As a result of GC analysis under<GC-1>, its purity was found to be 98% by area. The measurement resultsobtained were as follows.

¹H-NMR (400 MHz, CDCl₃) δ 1.42 (9H, s), 4.86 (2H, s)

[Example 3] Step (x): Production of Diethyl2-[(tert-Butylthio)methyl]-2-methylmalonate

Under nitrogen gas flow, 107 g (0.953 mol) of potassium-tert-butoxideand 790 g of tetrahydrofuran were placed in a 3-L reactor. To theresulting mixture, 158 g (0.907 mol) of diethyl methylmalonate was addeddropwise at an inner temperature within the range of 18° C. to 26° C.for 30 minutes. After stirring the resulting mixture for 30 minutes, 132g (0.953 mol) of tert-butylthiochloromethane produced in Example 2 wasadded dropwise thereto for 1 hour while the inner temperature was keptwithin the range of 18° C. to 26° C. After stirring the resultingmixture for 3 hours, 395 g of toluene and 395 g (0.229 mol) of 2 wt %hydrochloric acid were added thereto, followed by stirring the resultingmixture for 30 minutes. The aqueous layer was then removed byseparation. Subsequently, an operation of adding 395 g of water to thetoluene layer, stirring the resulting mixture for 1 hour, and thenremoving the aqueous layer by separation was repeated twice. Toluene wasremoved by concentrating the toluene layer by distillation. Thereafter,an operation of adding 250 g of ethanol to the resulting crude diethyl2-[(tert-butylthio)methyl]-2-methylmalonate, stirring the resultingmixture, and then removing the solvent by distillation was repeatedtwice. As a result, 246 g (0.848 mol; yield, 93.5%) of diethyl2-[(tert-butylthio)methyl]-2-methylmalonate was obtained. As a result ofGC analysis under <GC-1>, its GC purity was found to be 95.2% by area.The measurement results obtained were as follows.

¹H-NMR (400 MHz, CDCl₃) δ 1.26 (6H, t, J=7.1 Hz), 1.31 (9H, s), 1.48(3H, s), 3.03 (2H, s), 4.20 (4H, q, J=7.1 Hz)

[Reference Example 1] Production of Diethyl2-[(tert-Butylthio)methyl]malonate

Under nitrogen gas flow, 706 mg (23.5 mmol) of paraformaldehyde, 628 mg(3.15 mmol) of copper(I) acetate monohydrate, 1.32 g (22.0 mmol) ofacetic acid, 6.67 mL of toluene, and 3.00 g (18.7 mmol) of diethylmalonate were placed in a 100-mL three-necked flask, and a Dean-Starktube was attached to the flask, followed by stirring the resultingmixture at 110° C. for 6 hours. The reaction solution was allowed tocool to room temperature, and 4 mL of toluene was added thereto,followed by washing the organic layer twice with 10 mL of water and oncewith 10 mL of saturated aqueous sodium chloride solution. To theresulting solution, 1.00 mL (8.88 mmol) of tert-butyl mercaptan and 1.30mL (9.33 mmol) of triethylamine were added, and the resulting mixturewas stirred at 25° C. for 1.5 hours. This reaction solution was washedtwice with 6 mL of saturated aqueous ammonium chloride solution, andonce with 10 mL of saturated aqueous sodium chloride solution. Theobtained toluene layer was dried over anhydrous sodium sulfate, andsodium sulfate was removed by filtration. After removing toluene byconcentrating the filtrate by distillation, the obtained crude diethyl2-[(tert-butylthio)methyl]malonate was purified by silica gelchromatography, to obtain 1.32 g (5.02 mmol; yield, 26.8%) of diethyl2-[(tert-butylthio)methyl]malonate. The measurement results obtainedwere as follows.

¹H-NMR (400 MHz, CDCl₃) δ 1.28 (6H, t, J=7.1 Hz), 1.33 (9H, s), 3.03(2H, d, J=7.8 Hz), 3.51 (1H, t, J=7.8 Hz), 4.17-4.28 (4H, m)

[Example 4] Step (xi): Production of Diethyl2-[(tert-Butylthio)methyl]-2-methylmalonate

Under nitrogen gas flow, 3.50 mL of tetrahydrofuran and 0.160 g (4.00mmol) of 60 wt % sodium hydride were placed in a 50-mL three-neckedflask, and the flask was cooled on ice. To the resulting mixture, amixed solution of 0.700 g (2.67 mmol) of diethyl2-[(tert-butylthio)methyl]malonate produced in Reference Example 1 and3.50 mL of tetrahydrofuran was added dropwise. The resulting mixture wasstirred for 10 minutes, and a mixed solution of 0.183 mL (2.94 mmol) ofmethyl iodide and 2.00 mL of tetrahydrofuran was added dropwise to themixture for 15 minutes. To the resulting mixture, 0.0410 mL (0.659 mmol)of methyl iodide was added dropwise twice, and the resulting mixture wasstirred for 20 minutes. After adding 3.5 mL of water thereto, extractionwas performed twice with 7.0 mL of tert-butyl methyl ether. Theresulting layers of tert-butyl methyl ether were combined, and washedwith 3.5 mL of saturated brine, followed by separation. The obtainedtert-butyl methyl ether layer was concentrated to obtain 0.710 g of acolorless oily product. As a result of ¹H-NMR analysis, this oilyproduct was found to contain 0.646 g (2.34 mmol; yield, 87.6%) ofdiethyl 2-[(tert-butylthio)methyl]-2-methylmalonate.

[Example 5] Step (i): Production of Ethyl2-[(tert-Butylthio)methyl]-2-methylmalonate

Under nitrogen gas flow, 4.20 g (15.2 mmol) of diethyl2-[(tert-butylthio)methyl]-2-methylmalonate and 25 mL of ethanol wereplaced in a 100-mL recovery flask, and the flask was cooled to 2° C. Atan inner temperature within the range of 2° C. to 5° C., 16.0 mL (16.0mmol) of 1 mol/L aqueous sodium hydroxide solution was added dropwisethereto for 10 minutes. The resulting mixture was stirred at 22° C. for3 hours, and ethanol was then removed by distillation. The obtainedreaction solution was washed twice with 4.2 mL of toluene. To theaqueous layer, 17 mL of 1 mol/L hydrochloric acid was added to adjustthe pH to 1, and extraction was then performed 3 times with 4.2 mL oftoluene. The resulting toluene layers were combined, and dried over 4.20g of anhydrous sodium sulfate, followed by removing sodium sulfate byfiltration. Toluene was removed by concentrating the filtrate bydistillation, and the obtained crude ethyl2-[(tert-butylthio)methyl]-2-methylmalonate was purified by silica gelchromatography, to obtain 3.26 g of a colorless oily product. As aresult of ¹H-NMR analysis, this oily product was found to contain 3.16 g(12.7 mmol; yield, 83.5%) of ethyl2-[(tert-butylthio)methyl]-2-methylmalonate. The measurement resultsobtained were as follows.

¹H-NMR (400 MHz, CDCl₃) δ 1.29 (3H, t, J=7.1 Hz), 1.31 (9H, s), 1.54(3H, s), 3.02 (1H, d, J=11.9 Hz), 3.08 (1H, d, J=11.9 Hz), 4.24 (2H, q,J=7.1 Hz)

[Example 6] Step (ii): Production of Ethyl2-[(tert-Butylthio)methyl]-3-[(ethoxycarbonyl)oxy]-2-methyl-3-oxopropanoate

Under nitrogen gas flow, 2.21 g (8.90 mmol) of ethyl2-[(tert-butylthio)methyl]-2-methylmalonate produced in Example 5 and 28mL of toluene were placed in a 200-mL four-necked flask, and the flaskwas cooled to 0° C. To the resulting mixture, 1.36 mL (9.76 mmol) oftriethylamine was added dropwise for 2 minutes at an inner temperaturewithin the range of 1° C. to 8° C. Subsequently, 932 μL (9.79 mmol) ofethyl chloroformate was added dropwise thereto for 4 minutes at an innertemperature within the range of 1° C. to 8° C., and the resultingmixture was stirred at an inner temperature of 0° C. for 1 hour. As aresult of ¹H-NMR analysis, this reaction solution was found to containethyl2-[(tert-butylthio)methyl]-3-[(ethoxycarbonyl)oxy]-2-methyl-3-oxopropanoate.The measurement results obtained were as follows.

¹H-NMR (400 MHz, CDCl₃) δ 1.28 (3H, t, J=7.1 Hz), 1.32 (9H, s), 1.35(3H, t, J=7.1 Hz), 1.55 (3H, s), 3.03 (1H, d, J=12.1 Hz), 3.09 (1H, d,J=12.1 Hz), 4.24 (2H, q, J=7.1 Hz), 4.32 (2H, q, J=7.1 Hz)

[Example 7] Step (iii): Production of Ethyl2-(Azidocarbonyl)-2-[(tert-butylthio)methyl]propanoate

The solution containing ethyl2-[(tert-butylthio)methyl]-3-[(ethoxycarbonyl)oxy]-2-methyl-3-oxopropanoatein toluene, produced in Example 6 was cooled to 0° C., and an aqueoussolution prepared by mixing 908 mg (10.7 mmol) of sodium azide with 4.4mL of water was added to the above solution at an inner temperaturewithin the range of 0° C. to 3° C., followed by stirring the resultingmixture at an inner temperature of 0° C. for 6 hours. Subsequently, theaqueous layer was removed, and the obtained toluene layer was washedwith 10 mL of saturated aqueous sodium hydrogen carbonate solution, 10mL of saturated aqueous ammonium chloride solution, and 10 mL ofsaturated brine. The resulting toluene layers were combined, and driedover anhydrous sodium sulfate, followed by removing sodium sulfate byseparation. As a result of ¹H-NMR analysis, this solution was found tocontain ethyl 2-(azidocarbonyl)-2-[(tert-butylthio)methyl]propanoate.The measurement results obtained were as follows.

¹H-NMR (400 MHz, CDCl₃) δ 1.29 (3H, t, J=7.1 Hz), 1.31 (9H, s), 1.54(3H, s), 3.02 (1H, d, J=11.9 Hz), 3.08 (1H, d, J=11.9 Hz), 4.24 (2H, q,J=7.1 Hz)

[Example 8] Step (iv): Production of Ethyl2-[(tert-Butylthio)methyl]-2-isocyanatopropanoate

Under nitrogen gas flow, the solution containing ethyl2-(azidocarbonyl)-2-[(tert-butylthio)methyl]propanoate in toluene,produced in Example 7 was placed in a 300-mL four-necked flask, and thesolution was then stirred at an inner temperature within the range of75° C. to 80° C. for 3 hours, to obtain 23.1 g of a solution of ethyl2-[(tert-butylthio)methyl]-2-isocyanatopropanoate in toluene. As aresult of ¹H-NMR analysis, the content of ethyl2-[(tert-butylthio)methyl]-2-isocyanatopropanoate in this solution wasfound to be 1.65 g (6.72 mmol; yield, 75.5% from ethyl2-[(tert-butylthio)methyl]-2-methylmalonate). The measurement resultsobtained were as follows.

¹H-NMR (400 MHz, CDCl₃) δ 1.29 (3H, t, J=7.1 Hz), 1.30 (9H, s), 1.52(3H, s), 2.78 (1H, d, J=12.1 Hz), 2.98 (1H, d, J=12.1 Hz), 4.23 (2H, q,J=7.1 Hz)

[Example 9] Step (vi-1): Production of Ethyl4-Methyl-2-oxo-1,3-thiazolidine-4-carboxylate

To 6.01 g of the solution containing 353 mg (1.44 mmol) of ethyl2-[(tert-butylthio)methyl]-2-isocyanatopropanoate in toluene, producedin Example 8, 288 mg (1.51 mmol) of p-toluenesulfonic acid monohydratedissolved in 1.30 mL of acetone was added, and the resulting mixture wasstirred at an inner temperature of 20° C. for 2.5 hours. Subsequently,the solvent was concentrated, and 1.4 mL of water was added thereto. ThepH was adjusted to 7 by addition of 1 mol/L aqueous sodium hydroxidesolution. After carrying out extraction 4 times with 1.1 mL of tolueneand twice with 1.1 mL of tert-butyl methyl ether, the resulting organiclayers were combined and concentrated, to obtain a pale yellow oilyproduct. As a result of ¹H-NMR analysis, this oily product was found tocontain ethyl 4-methyl-2-oxo-1,3-thiazolidine-4-carboxylate. Themeasurement results obtained were as follows.

¹H-NMR (400 MHz, CDCl₃) δ 1.32 (3H, t, J=7.1 Hz), 1.61 (3H, s), 3.30(1H, d, J=11.4 Hz), 3.82 (1H, d, J=11.4 Hz), 4.27 (2H, q, J=7.1 Hz)

[Example 10] Step (vi-2) and Step (vii): Production of4-Methyl-2-oxo-1,3-thiazolidine-4-carboxylic Acid and α-MethylcysteineHydrochloride

Under nitrogen gas flow, the oily product containing ethyl4-methyl-2-oxo-1,3-thiazolidine-4-carboxylate produced in Example 9 wasplaced in a 50-mL recovery flask, and 9.4 mL (56 mmol) of 6 mol/Lhydrochloric acid was added thereto, followed by stirring the resultingmixture at an inner temperature of 100° C. for 10 hours. As a result of¹H-NMR analysis of this reaction solution, the reaction solution wasfound to contain 4-methyl-2-oxo-1,3-thiazolidine-4-carboxylic acid.

¹H-NMR (400 MHz, D₂O) δ 1.52 (3H, s), 3.40 (1H, d, J=11.6 Hz), 3.69 (1H,d, J=11.6 Hz)

Subsequently, this reaction solution was stirred at an inner temperatureof 100° C. for additional 30 hours. As a result of ¹H-NMR analysis ofthis reaction solution, the reaction solution was found to containα-methylcysteine hydrochloride. The measurement results obtained were asfollows.

¹H-NMR (400 MHz, D₂O) δ 1.53 (3H, s), 2.84 (1H, d, J=15.0 Hz), 3.11 (1H,d, J=15.0 Hz)

[Example 11] Cloning of Hydrolase Genes for Step (i)

Based on a gene sequence (cenp, SEQ ID NO:1) encoding carboxyesterase NP(CENP, GenBank Accession No. AAC43262, SEQ ID NO:2), which is ahydrolase derived from the Bacillus subtilis Thail-8 strain, primers foramplifying the entire cenp gene, npfw (SEQ ID NO:19) and npry (SEQ IDNO:20) were designed and synthesized. PCR was carried out according to aconventional method using, as a template, chromosomal DNA of each of theBacillus subtilis NBRC3108 strain, Bacillus subtilis NBRC3215 strain,Bacillus subtilis NBRC3335 strain, Bacillus subtilis NBRC14144 strain,Bacillus subtilis NBRC14191 strain, Bacillus subtilis NBRC14473 strain,Bacillus subtilis NBRC101246 strain, and Bacillus subtilis NBRC101590strain, and the primers npfw (SEQ ID NO:19) and npry (SEQ ID NO:20). Asa result, a total of eight kinds of DNA fragments each having a lengthof about 1.0 kbp were obtained.

Each of the eight kinds of DNA fragments obtained was digested withEcoRI and XbaI. According to a conventional method, each of theresulting DNA fragments was introduced downstream of the trc promoter ofMunI/XbaI-digested pKW32, which is a plasmid described inPCT/JP2011/069680, to obtain plasmids pKCENP1, pKCENP2, pKCENP3,pKCENP4, pKCENP5, pKCENP6, pKCENP7, and pKCENP8.

The sequence of the hydrolase gene inserted in each of the total ofeight kinds of plasmids obtained was identified according to aconventional method. The gene sequences were cenp-1 (SEQ ID NO:3),cenp-2 (SEQ ID NO:5), cenp-3 (SEQ ID NO:7), cenp-4 (SEQ ID NO:9), cenp-5(SEQ ID NO:11), cenp-6 (SEQ ID NO:13), cenp-7 (SEQ ID NO:15), and cenp-8(SEQ ID NO:17), respectively. The amino acid sequences of the hydrolasesencoded by the genes were CENP-1 (SEQ ID NO:4), CENP-2 (SEQ ID NO:6),CENP-3 (SEQ ID NO:8), CENP-4 (SEQ ID NO:10), CENP-5 (SEQ ID NO:12),CENP-6 (SEQ ID NO:14), CENP-7 (SEQ ID NO:16), and CENP-8 (SEQ ID NO:18),respectively.

Using the eight kinds of plasmids obtained, E. coli JM109 (manufacturedby Takara Bio Inc.) was transformed according to a conventional method,to obtain recombinant E. coli JM109/pKCENP1, JM109/pKCENP2,JM109/pKCENP3, JM109/pKCENP4, JM109/pKCENP5, JM109/pKCENP6,JM109/pKCENP7, and JM109/pKCENP8. In order to obtain bacterial cellsexpressing these genes, each recombinant E. coli was cultured in liquidLB medium supplemented with kanamycin and a lac promoter inducer at 30°C. The cells were collected after about 20 hours of the culture.

Among the hydrolases obtained, homology between the amino acid sequenceCENP-1 (SEQ ID NO:4) and each of the other amino acid sequences (SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18) was calculated using BLAST. The results are shownin Table 1.

Homology between the hydrolase amino acid sequence CENP-1 (SEQ ID NO:4)and the amino acid sequence of each of commercially available enzymes,Bacillus licheniformis-derived protease (commercially available enzyme,manufactured by Sigma-Aldrich) and pig liver-derived esterase(commercially available enzyme, manufactured by Sigma-Aldrich), wasinvestigated by searching by BLAST. As a result, no homology was found.

TABLE 1 Homology to Bacillus Amino acid subtilis IFO3108- sequence ofSEQ ID derived carboxyesterase hydrolase NO NP (SEQ ID NO: 4) CENP-1 4100%  CENP-2 6 99% CENP-3 8 99% CENP-4 10 99% CENP-5 12 99% CENP-6 1499% CENP-7 16 99% CENP-8 18 99%

[Example 12] Screening for Hydrolase for Step (i)

In an aqueous solution containing 100 mmol/L potassium phosphate, cellsof each of the eight kinds of bacteria obtained in Example 11 and 30 g/Ldiethyl 2-[(tert-butylthio)methyl]-2-methylmalonate were mixed, and thereaction was allowed to proceed at 30° C. at pH 7 overnight withshaking. Bacillus licheniformis-derived protease (manufactured bySigma-Aldrich) was also allowed to react under the same conditionsovernight with shaking.

To 200 μL of each solution after the reaction, 100 μL of 1 mol/Lhydrochloric acid was added to stop the reaction, and the reactionsolution was then centrifuged at 10,000 rpm. After mixing 50 μL of thesupernatant obtained after the centrifugation with 50 vol % acetonitrileand 950 μL of 50 mmol/L trifluoroacetic acid solution, the opticalpurity was analyzed under the conditions of <HPLC-2>.

Bacterial cells of each of commercially available enzymes, Bacillusstearothermophilus-derived esterase BS1 (manufactured by Julich FineChemicals), Bacillus stearothermophilus-derived esterase BS3(manufactured by Julich Fine Chemicals), or pig liver-derived esterase(manufactured by Sigma-Aldrich), were allowed to react with 5 g/Ldiethyl 2-[(tert-butylthio)methyl]-2-methylmalonate in an aqueoussolution containing 100 mmol/L potassium phosphate at 30° C. at pH 7overnight with shaking. To 250 μL of the solution after the reaction,250 μL of acetonitrile was added, and the resulting mixture wascentrifuged at 10,000 rpm. The resulting centrifugation supernatant wasanalyzed under the conditions of <HPLC-2>, to investigate the opticalpurity of the ethyl 2-[(tert-butylthio)methyl]-2-methylmalonateproduced.

These results are shown in Table 2 and FIG. 2.

TABLE 2 Optical purity of (S)-isomer Name of recombinant E. coli orenzyme ee (%) JM109/pKCENP1 99.8 JM109/pKCENP2 99.7 JM109/pKCENP3 99.7JM109/pKCENP4 99.8 JM109/pKCENP5 99.8 JM109/pKCENP6 99.8 JM109/pKCENP799.6 JM109/pKCENP8 99.8 Bacillus licheniformis-derived protease(commercially 94.5 available enzyme, manufactured by Sigma-Aldrich)Bacillus stearothermophilus-derived esterase BS1 −95.4 (commerciallyavailable enzyme, manufactured by Julich Fine Chemicals) Bacillusstearothermophilus-derived esterase BS3 −95.4 (commercially availableenzyme, manufactured by Julich Fine Chemicals) Pig liver-derivedesterase (commercially available enzyme, −84.3 manufactured bySigma-Aldrich)

[Example 13] Step (i): Production of Ethyl(S)-2-[(tert-Butylthio)methyl]-2-methylmalonate

In a 1-L jar fermenter (manufactured by ABLE Corporation, Type BMJ),14.4 g (52.1 mmol) of diethyl2-[(tert-butylthio)methyl]-2-methylmalonate, 65.0 g of 1 mol/L phosphatebuffer (pH 7), 26.0 g of the recombinant E. coli JM109/pKCENP1 preparedin Example 11, and 555 g of desalted water were fed, and the resultingmixture was sufficiently mixed. Thereafter, the reaction was allowed toproceed at 30° C. at 500 rpm for 16 hours. During the reaction, the pHwas kept at 7.0 using 20 wt % aqueous sodium hydroxide solution.Completion of the reaction was judged by analysis under the conditionsof <HPLC1>, based on disappearance of diethyl2-[(tert-butylthio)methyl]-2-methylmalonate.

The obtained reaction solution was centrifuged at 8000 rpm, to obtain areaction solution supernatant. Thereafter, the supernatant was subjectedto treatment with a microfiltration membrane having a pore size of 0.2μm for removal of the bacterial cells, and further to treatment with anultrafiltration membrane with a molecular weight cutoff of 10,000. Tothe obtained filtrate, 160 mL of toluene was added, and the resultingmixture was sufficiently stirred followed by separation, therebyremoving residual impurities such as diethyl2-[(tert-butylthio)methyl]-2-methylmalonate into the organic layer, andobtaining an aqueous layer containing ethyl2-[(tert-butylthio)methyl]-2-methylmalonate. The pH of the aqueous layerwas lowered to 2.0 using 6 mol/L sulfuric acid, and 160 mL of toluenewas added to the solution, followed by sufficiently stirring theresulting mixture, thereby extracting ethyl2-[(tert-butylthio)methyl]-2-methylmalonate into the toluene layer. Byadding 160 mL of toluene to the remaining aqueous layer, andsufficiently mixing the resulting mixture, residual ethyl2-[(tert-butylthio)methyl]-2-methylmalonate in the aqueous layer wasalso extracted into the toluene layer.

As a result of analysis of the obtained toluene solution under theconditions of

<HPLC1>, it was found that 11.7 g (47.1 mmol; yield, 90.4%) of ethyl2-[(tert-butylthio)methyl]-2-methylmalonate was obtained. Themeasurement results obtained were as follows.

¹H-NMR (400 MHz, CDCl₃) δ 1.29 (3H, t, J=7.1 Hz), 1.31 (9H, s), 1.54(3H, s), 3.02 (1H, d, J=11.9 Hz), 3.08 (1H, d, J=11.9 Hz), 4.24 (2H, q,J=7.1 Hz)

As a result of analysis of the optical purity under the conditions of<HPLC2>, the configuration of the ethyl2-[(tert-butylthio)methyl]-2-methylmalonate obtained was found to be(S), and its optical purity was found to be 99.8% e.e.

[Example 14] Step (ii): Production of Ethyl(R)-2-[(tert-Butylthio)methyl]-3-[(ethoxycarbonyl)oxy]-2-methyl-3-oxopropanoate

Under nitrogen gas flow, 22.4 g (90.1 mmol) of ethyl(S)-2-[(tert-butylthio)methyl]-2-methylmalonate produced in Example 13and 130 mL of toluene were placed in a 300-mL four-necked flask. Theresulting mixture was cooled to 5° C., and 10.0 g (99.1 mmol) oftriethylamine was added dropwise to the mixture at an inner temperaturewithin the range of 5° C. to 10° C. for 5 minutes. Under nitrogen gasflow, 10.8 g (99.2 mmol) of ethyl chloroformate and 22.4 mL of toluenewere placed in a 500-mL four-necked flask, and the resulting mixture wascooled to 5° C. To this mixture, the solution of triethylamine salt ofethyl (S)-2-[(tert-butylthio)methyl]-2-methylmalonate in toluenepreliminarily prepared was added dropwise at an inner temperature withinthe range of 5° C. to 12° C. for 15 minutes. Subsequently, the resultingmixture was stirred for additional 1 hour while an inner temperature of5° C. was kept. As a result of ¹H-NMR analysis, this solution in toluenewas confirmed to be a solution containing ethyl(R)-2-[(tert-butylthio)methyl]-3-[(ethoxycarbonyl)oxy]-2-methyl-3-oxopropanoate.The measurement results obtained were as follows.

¹H-NMR (400 MHz, CDCl₃) δ 1.28 (3H, t, J=7.1 Hz), 1.32 (9H, s), 1.35(3H, t, J=7.2 Hz), 1.55 (3H, s), 3.03 (1H, d, J=12.1 Hz), 3.09 (1H, d,J=12.1 Hz), 4.24 (2H, q, J=7.1 Hz), 4.32 (2H, q, J=7.1 Hz)

[Example 15] Step (iii): Production of Ethyl(S)-2-(azidocarbonyl)-2-[(tert-butylthio)methyl]propanoate

The solution of ethyl(R)-2-[(tert-butylthio)methyl]-3-[(ethoxycarbonyl)oxy]-2-methyl-3-oxopropanoatein toluene produced in Example 14 was cooled to 2° C., and an aqueoussolution prepared by mixing 9.19 g (108 mmol) of sodium azide with 40 mLof water was added dropwise to the solution at an inner temperaturewithin the range of 2° C. to 4° C. for 4 minutes. The resulting mixturewas stirred at an inner temperature of 2° C. for 1 hour, and 60 mL (60.0mmol) of 1 mol/L hydrochloric acid was added dropwise to the mixture atan inner temperature within the range of 2° C. to 3° C. for 10 minutes.After separating the mixture into a toluene layer and an aqueous layer,the aqueous layer was subjected to extraction again by addition of 45 mLof toluene, and the resulting toluene layer was combined with thepreviously obtained toluene layer. The combined toluene layer was washedwith 35 mL of 5 wt % aqueous sodium hydrogen carbonate solution. To thetoluene layer obtained, 11.2 g of anhydrous magnesium sulfate was added,and the resulting mixture was stirred at 0° C. for 2 hours, followed bycarrying out filtration. The magnesium sulfate separated by thefiltration was washed with 11 mL of toluene, and the washing liquid wasmixed with the filtrate. As a result of ¹H-NMR analysis of the obtainedsolution in toluene, the solution in toluene was found to contain ethyl(S)-2-(azidocarbonyl)-2-[(tert-butylthio)methyl]propanoate. Themeasurement results obtained were as follows.

¹H-NMR (400 MHz, CDCl₃) δ 1.29 (3H, t, J=7.1 Hz), 1.31 (9H, s), 1.54(3H, s), 3.02 (1H, d, J=11.9 Hz), 3.08 (1H, d, J=11.9 Hz), 4.24 (2H, q,J=7.1 Hz)

[Example 16] Step (iv): Production of Ethyl(S)-2-[(tert-Butylthio)methyl]-2-isocyanatopropanoate

Under nitrogen gas flow, 45 mL of toluene was placed in a 500-mLfour-necked flask, and heated to 85° C. To the flask, the solutioncontaining 23.3 g (85.3 mmol) of ethyl(S)-2-(azidocarbonyl)-2-[(tert-butylthio)methyl]propanoate in tolueneproduced in Example 15 was added dropwise at an inner temperature withinthe range of 84° C. to 91° C. for 4 hours. Thereafter, the resultingmixture was stirred at 85° C. for 1 hour, to obtain a solution of ethyl(S)-2-[(tert-butylthio)methyl]-2-isocyanatopropanoate in toluene. As aresult of ¹H-NMR analysis, the amount of ethyl(S)-2-[(tert-butylthio)methyl]-2-isocyanatopropanoate contained in thissolution in toluene was found to be 21.5 g (87.6 mmol; yield from ethyl(S)-2-[(tert-butylthio)methyl]-2-methylmalonate, 97.2%). The measurementresults obtained were as follows.

¹H-NMR (400 MHz, CDCl₃) δ 1.29 (3H, t, J=7.1 Hz), 1.30 (9H, s), 1.52(3H, s), 2.78 (1H, d, J=12.1 Hz), 2.98 (1H, d, J=12.1 Hz), 4.23 (2H, q,J=7.1 Hz)

[Example 17] Step (vi-1) and Step (vi-2): Production of Ethyl(S)-4-Methyl-2-oxo-1,3-thiazolidine-4-carboxylate and(S)-4-Methyl-2-oxo-1,3-thiazolidine-4-carboxylic Acid

Under nitrogen gas flow, 39.2 g of a solution containing 11.2 g (45.6mmol) of ethyl (S)-2-[(tert-butylthio)methyl]-2-isocyanatopropanoate intoluene was placed in a 200-mL four-necked flask. To the flask, 9.19 g(88.4 mmol) of 35 wt % hydrochloric acid was added, and the resultingmixture was stirred at 25° C. for 45 minutes. As a result of ¹H-NMRanalysis of the toluene layer of this reaction solution, the layer wasfound to contain ethyl(S)-4-methyl-2-oxo-1,3-thiazolidine-4-carboxylate.

¹H-NMR (400 MHz, CDCl₃) δ 1.32 (3H, t, J=7.1 Hz), 1.60 (3H, s), 3.28(1H, d, J=11.4 Hz), 3.80 (1H, d, J=11.4 Hz), 4.26 (2H, q, J=7.1 Hz)

Subsequently, the inner temperature of this reaction solution wasincreased to 60° C. to 70° C., and the reaction solution was thenstirred for additional 1 hour. After removing toluene by distillation,22.6 g of water and 2.40 g (23.0 mmol) of 35 wt % hydrochloric acid wereadded to the solution, and the resulting mixture was stirred at an innertemperature within the range of 70° C. to 90° C. for 4 hours. Afterallowing the reaction solution to cool to room temperature, precipitatedcrystals were collected by filtration. The obtained crystals were washedwith 23 mL of toluene and 23 mL of water, to obtain 5.97 g of whitecrystals. As a result of HPLC analysis under the conditions of <HPLC-3>,the white crystals were found to contain 5.56 g (34.5 mol; yield, 75.7%)of (S)-4-methyl-2-oxo-1,3-thiazolidine-4-carboxylic acid. Themeasurement results on the compound obtained were as follows.

¹H-NMR (400 MHz, D₂O) δ 1.52 (3H, s), 3.40 (1H, d, J=11.6 Hz), 3.69 (1H,d, J=11.6 Hz)

[Example 18] Step (vi-1) and Step (vi-2): Production of Ethyl(S)-4-Methyl-2-oxo-1,3-thiazolidine-4-carboxylate and(S)-4-Methyl-2-oxo-1,3-thiazolidine-4-carboxylic Acid

Under nitrogen gas flow, 4.62 g of a solution containing 1.33 g (5.4mmol) of ethyl (S)-2-[(tert-butylthio)methyl]-2-isocyanatopropanoate intoluene was placed in a 50-mL test tube. To the test tube, 1.52 g (5.4mmol) of p-toluenesulfonic acid monohydrate was added, and the resultingmixture was stirred at room temperature for 60 minutes, followed byadding 8 ml (16.0 mmol) of 2 mol/L aqueous sodium hydroxide solutionthereto at the same temperature. After stirring the resulting mixturefor 1.5 hours, the toluene layer was removed by separation. To theobtained aqueous layer, 0.4 g of 98 wt % sulfuric acid was added at roomtemperature. As a result, precipitation of crystals occurred. Themixture was cooled to an inner temperature of 5° C., and theprecipitated crystals were collected by filtration. The obtainedcrystals were washed with a small amount of water, to obtain 0.70 g (4.3mmol; yield, 79.6%) of (S)-4-methyl-2-oxo-1,3-thiazolidine-4-carboxylicacid.

[Example 19] Step (vii): Production of α-Methyl-D-cysteine Hydrochloride

Under nitrogen gas flow, 4.00 g of a white solid containing 3.72 g (23.1mmol) of (S)-4-methyl-2-oxo-1,3-thiazolidine-4-carboxylic acid producedin Example 17 was placed in a 100-mL four-necked flask. To the flask, 60mL (720 mmol) of 35 wt % hydrochloric acid was added, and the resultingmixture was heated to an inner temperature of 100° C., followed bystirring the mixture for 51 hours. After removing 35 wt % hydrochloricacid by distillation, an operation of adding 5 mL of toluene andremoving 35 wt % hydrochloric acid by azeotropic distillation wasrepeated twice. To the solid of crude α-methyl-D-cysteine hydrochlorideobtained, 12 mL of 2-propanol was added, and the solid was dissolved byheating at 50° C. To the resulting solution, 20 mL of toluene was added,and 8.56 g of the solvent was removed by distillation. The remainingsolution was cooled to 0° C. for 2 hours, and the crystals produced werecollected by filtration. The crystals obtained were washed with 4 mL oftoluene, and dried at 20° C. for 1.5 hours under reduced pressure, toobtain 2.89 g of white crystals. As a result of HPLC analysis under theconditions of <HPLC-3>, the white crystals were found to contain 2.71 g(yield, 68.4%) of α-methyl-D-cysteine hydrochloride. Based on the resultof HPLC analysis under the conditions of <HPLC-4>, the optical puritywas 99.9% e.e. The measurement results on the compound obtained were asfollows.

¹H-NMR (400 MHz, D₂O) δ 1.53 (3H, s), 2.84 (1H, d, J=15.0 Hz), 3.11 (1H,d, J=15.0 Hz)

INDUSTRIAL APPLICABILITY

By the present invention, an optically active α-substituted cysteine ora salt thereof, which is useful as an intermediate for pharmaceuticalsand the like, can be produced more simply, quickly, and safely, using aneasily available and inexpensive material. The present invention enablesstable production of the optically active α-substituted cysteine or thesalt thereof in an industrial scale.

1. A method for producing an α-substituted cysteine represented byGeneral Formula (1):

wherein R² represents a C₁-C₄ alkyl group or a salt thereof, said methodcomprising the steps of: (a) converting, by Curtius rearrangementreaction, a compound represented by General Formula (5):

wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² has the samemeaning as R² in the General Formula (1), to obtain a compoundrepresented by General Formula (6):

wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (5), and (b) subjecting said compound represented by GeneralFormula (6) to a process of converting the isocyanate group to an aminogroup, a process of hydrolyzing the ester group, and a process ofremoving the tert-butyl group by action of an acid.
 2. The method ofclaim 1, wherein said step (b) comprises the steps of: (b-1) allowing anacid to act on a compound represented by General Formula (6):

wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² has the samemeaning as R² in the General Formula (1), to construct a thiazolidinonering, for conversion into a compound represented by General Formula(7-1):

wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (6); (b-2) allowing an acid or a base to act on said compoundrepresented by the General Formula (7-1) to hydrolyze the ester group,to obtain a compound represented by General Formula (7-2):

wherein R² has the same meaning as R² in the General Formula (7-1); and(c) allowing an acid or a base to act on said compound represented bythe General Formula (7-2) to open the thiazolidinone ring, to producesaid α-substituted cysteine represented by the General Formula (1) orsalt thereof.
 3. A method for producing an α-substituted cysteinerepresented by General Formula (1):

wherein R² represents a C₁-C₄ alkyl group or a salt thereof, said methodcomprising the steps of: (a) allowing an acid or a base to act on acompound represented by General Formula (7-1):

wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² has the samemeaning as R² in the General Formula (1) to hydrolyze the ester group,to obtain a compound represented by General Formula (7-2):

wherein R² has the same meaning as R² in the General Formula (7-1); and(b) allowing an acid or a base to act on said compound represented byGeneral Formula (7-2) to open the thiazolidinone ring.
 4. The method ofclaim 3, said method comprising the step of: (c) allowing an acid to acton a compound represented by General Formula (6):

wherein R¹ represents a C₁-C₁₀ alkyl group which is optionallysubstituted, a C₇-C₂₀ aralkyl group which is optionally substituted, ora C₆-C₁₂ aryl group which is optionally substituted, and R² has the samemeaning as R² in the General Formula (1) to construct a thiazolidinonering, to obtain a compound represented by General Formula (7-1):

wherein R¹ and R² have the same meanings as R¹ and R² in the GeneralFormula (6).